© 2015 Nature America, Inc. All rights reserved. Sciences, Sciences, Beijing, China. Central China Normal University, Wuhan, China. University of Science and Technology of China, Hefei, China. 1 and CDR1as functions, microRNA regulating in roles their with Sry sponges, microRNA as a as CDR1as with for sponge miR-7 from the and circRNA generated function to able are circRNAs two least at noncoding probably roles latory specific and tissue-specific expression, thus suggesting potential regu cells animal in members of thousands with RNAs of species been large a now as have recognized circRNAs analysis, bioinformatic and sequencing sporadically reported been have genes coding specific of exons from generated circRNAs then ago decades three than more EM by indicated was cells both with functions gene-regulatory their execute that ncRNA-a, as such ncRNAs, also affect the expression of only genes limited neighboring ncRNAs such as linc-HOXA1 and Air function in it is transcribed where locus away the from located of genes in transcription gene regulate complex transcription II) (Pol II polymerase RNA the with association their via transcription modulate RNA 7SK and RNA U1 as such ncRNAs status of chromatin modification epigenetic the through sion Multiple long ncRNAs such as XIST and HOTAIR control gene expres expression gene regulate to is of ncRNAs roles central of the One interaction expression genes. EIciRNAs. In microRNA gene Noncoding Huijie Liu Bin Yu Zhaoyong Li in the nucleus Exon-intron circular RNAs regulate transcription nature structural & molecular biology molecular & structural nature Received 10 November 2014; accepted 19 December 2014; published online 9 February 2015; School School of Life Sciences, University of Science and Technology of China, Hefei, China.

Several lines of evidence have indicated that circRNAs are most most are circRNAs that indicated have evidence of lines Several mammalian in linkages covalent with circRNAs of existence The these gene (circSry) as a for (refs. sponge miR-138 (circSry) gene

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and been col was not optimized for detecting circRNAs; second, for col detecting was not optimized proto the deep-sequencing first, for two reasons: be underestimated might data deep-sequencing the from deduced of circRNAs number read number was low for most circRNAs identified. We believe that the We noted that, similarly to data described in a previous report a previous in described data to We similarly that, noted 1 Tables ( cells HeLa in Pol II with associated were circRNAs some that revealed analysis bioinformatic subsequent and samples ( II Pol to antibody an with (CLIP) immunoprecipitation and linking regulation and sought to Polidentify ncRNAs II–associated via cross- transcriptional in involved are ncRNAs some that Wehypothesized A RESULTS in genes parental RNA-RNA interaction. their of transcription II Pol regulate the circRNAs these of some that evidence of the lines provide in we localize circRNAs these characterizations, and molecular that nucleus. Withbiochemistry further found we and undescribed, previously was knowledge, our to that, circRNAs of subclass a fied identi We transcription. gene regulate might RNAs these of some cytoplasm sponges microRNA as the function in not may thus and microRNAs for sites binding multiple possess not localized do circRNAs of majority the that shown has predominantly analysis Bioinformatic are circSry

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© 2015 Nature America, Inc. All rights reserved. intronic sequences when examined by RT-PCR with divergent primers 2a Fig. ( cases both in retained were exons ized ( circRNAs, circEIF3J and circPAIP2, by northern blotting and RT-PCR methodology described viously (RT-PCR; PCR reverse-transcription divergent and digestion R RNase by circular were they that firmed ( cells HEK293 such as expression in circCLTC, HeLa and cell-specific demonstrated PCR ( RNA II Pol immunoprecipitation by (RNA circRNAs IP) and subsequent abundant real-time quantitative most 15 the of enrichment the missing all other within enclosed sequences circRNAs. We confirmed thus reads, as circRNA sequences junction only count could analysis EIciRNA. the in sequences intron the to complementary 5 (the EIciRNA the in sequences exonic the to complementary PAIP2 for Immunostaining cells. HEK293 of nuclei the to circRNAs both of localization showing circPAIP2, and circEIF3J in shown are blots northern of images Uncropped nt. 1,000 K denotes plasmids. circRNA-overexpression the with transfected cells from RNA O.E., probes. intron and exon with hybridization of ( experiments. triplicate from and hsa-circRNA2 R digestion. RNase to circFUNDC1 and ( marker. size M, cells. HeLa in circRNAs 12 other the of circularization showing primers ( transcription. RT, DNA; genomic reverse gDNA, control; control; positive circRNA known hsa-circRNA2, primers. convergent and divergent using circRNAs, are circFUNDC1 ( EIciRNAs. identify to sequencing RNA by followed ( EIciRNA. 1 Figure s e l c i t r a  5 the to corresponding d Amplicon (bp) c a b Fig. 1b Fig. 100 bp 100 bp 200 bp 400 bp 600 300 bp 300 bp 500

) Verification that circEIF3J, circPAIP2 and and circPAIP2 circEIF3J, that ) Verification Relative RNA level HeLa ci 0. 1. 1. 2. rc EIF3J 0 5 0 5 0 mRNA is shown for comparison. Scale bars, 10 10 bars, Scale comparison. for shown is mRNA

Table ci r RNA sequencing ). In actuality, all 15 circRNAs analyzed appeared to contain contain to appeared analyzed circRNAs 15 all actuality, In ).

cPAIP2 RNA isolation

ci

, r Identification and characterization of characterization and Identification

cFUNDC1 M e

a hsa-circRNA2 ). The data demonstrated that introns between circular between introns that demonstrated data The ). Pol II ) Experimental process for Pol II CLIP CLIP II Pol for process ) Experimental 175 1 Cross-lin Reverse cross-link Reverse CLIP withanti–PolII and

c circRSRC1 ) RT-PCR products with divergent divergent with ) RT-PCR products Supplementary Fig. 1f Fig. Supplementary

GAPDH 173 HeLa circMIER1 Supplementary Fig. 1b Supplementary EIF3J k ACTB 100 circSSR1 PAIP2 mRNA RNase RNase 108 β

′ circWDR60 -actin mRNA, negative negative mRNA, -actin mRNA exon and upstream intron closest to the the to closest intron upstream and exon R R 167 e + – ) Northern blots of of blots ) Northern circRBM33 152 b circMAN1A2-1 β hsa-circRNA2 ci ci ci 2

Relative RNA level -actin rc rc rc 4 c 0. 1. 1. 2. . We further analyzed two of these these of two analyzed We . further FUNDC1 PAIP2 ir EIF3J 109 c 0 5 0 5 0 EIF3J circMAN1A2-2 ci rcPAIP2

ci 108 Fig. 1b Fig. rcFUNDC1

). For the 15 RNAs, we con we RNAs, 15 the For ). hsa-circRNA2 circNAP1L4 Fig. 1 Fig. HeLa 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp 192 –

e circBPTF

). ). Some of the circRNAs, HEK293 144 EIF3J Supplementary Data Set 1 Set Data Supplementary –

GAPDH

e circMAN1A2-3

d and and No No ), according to pre to according ),

EIF3J 150 ACTB

µ circCLTC RT RNase RNase and and

m. Split-channel images are shown in in shown are images Split-channel m. PAIP2 mRNA Supplementary Supplementary 100 cD f circCDK11B GAPDH mRNA Junction probe PAIP2 R R NA + – ′ exon), junction probe is complementary to the junction sequences, and intron probe is probe intron and sequences, junction the to complementary is probe junction exon), advance online publication online advance gD NA , , transcripts showing that both circEIF3J and circPAIP2 are EIciRNAs, on the basis basis the on EIciRNAs, are circPAIP2 and circEIF3J both that showing transcripts e Exon EIF3J (55 nt) ACTB RNase circEIF3J circEIF3J circEIF3J (500 nt) Intron 647 nt 3 mRNA 101 bp 130 bp 170 bp 125 bp 180 bp 166 bp 199 bp 115 bp 187 bp , , ­ - - Exon (92 nt) rRNAs R

EIF3J 3 . . ( Intron probe 4 gest that they might be involved in transcriptional regulation. transcriptional in involved be might they that gest sug Pol II with association their and EIciRNAs these of localization ( nucleus the in (FISH) revealed that circEIF3J and circPAIP2 are exclusivelylocalized (circPAIP2/ ~9.4% (circEIF3J/ ~8.9% is mRNA parental to EIciRNA of ratio the that calculated also we blots, northern From Methods). in Online analysis (copy-number in and respectively, HeLa cells ~22, approximate and circEIF3J circPAIP2 copy number per cell to be ~31 the We estimated EIciRNAs. circRNAs intron-containing identified exclusively of exonic sequences consist they but back-splicing, exon from formed also are circRNAs property of these circRNAs is distinct from microRNA-sponge as hsa-circRNA6, microRNA-sponge hsa-circRNA2 and hsa-circRNA9 (refs. from distinct is circRNAs and the other circRNAs experimentally, such characterized circRNAs these of property 3 f ) Representative FISH images ( images FISH ) Representative ′ exon in the circRNA ( 12.5 and and – β hsa-circRNA ci ci ci -actin r rc rc Exon prob cFUNDC1 25 PAIP2 EIF3 – Endogenous PAIP2 12.5 J d + ) Real-time PCR showing resistance of circEIF3J, circPAIP2 circPAIP2 circEIF3J, of resistance showing PCR ) Real-time I e 2 EIF3J HEK293 mRNAs are positive or negative controls. Error bars, s.e.m. s.e.m. bars, Error controls. negative or positive are mRNAs 25 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp 100 bp 200 bp + Supplementary Figure 2c Figure Supplementary Fig. 1 Fig. ntron probe mRNA RNA 25 + PAIP2

O.E. No No nature structural & molecular biology molecular & structural nature + 1 f RT and and Supplementary Fig. 2b Acti (μg) 0.2 1.0 1.5 2.0 3.0 0.5k mRNA). Fluorescence mRNA). Fluorescence cD n Junction probe k k k k k NA Supplementary Fig. 2c Fig. Supplementary PAIP2 n (164 nt) 2 Exon gD = 150 cells for each circRNA) of circRNA) each for cells = 150 DAPI 7 RNase . We have therefore termed our newly circPAIP2 NA circPAIP2 (641 nt Intron 985 nt mRNA 2 β circPAIP2 -actin and FISH for for FISH and -actin (180 nt Exon rRNA R 2 ) . For . For 101 bp 130 bp 170 bp 125 bp 180 bp 166 bp 199 bp 115 bp 187 bp 3 s ) 12.5 Intron prob – e and and Exon probe 25 – Endogenou ). The intron Exo in in situ f 12. , exon probe is probe , exon n EIF3J + e 5 Convergent primers Divergent primers – Exon g 25 + ). The nuclear nuclear The ). hybridization hybridization O s EIF3J mRNA) and and mRNA) Intron probe PAIP2 27 25 + Exo , - 2 retention and and

8 mRNA

+ n 5 .E ); ); these . Exo ( 0.2k 0.5k 1.0k 1.5k 2.0k 3.0k µ

g) n - © 2015 Nature America, Inc. All rights reserved. transfections. * transfections. In ( cells. ( Methods. Online in are plasmids overexpression the about Details cells. HeLa in constructs various with circPAIP2 ( counted. were region flanking the of kb 1.5 within elements complementary two the of part one least at had that repeats complementary those Only labeled. are circRNAs forming exons the to sequences repeat the from distances linear The element. Alu an than other sequences complementary flanking contain four another and pairs, complementary Alu flanking contain circRNAs 15 of 4 out of ( 2 Figure for ~33-fold and circEIF3J for ~548-fold of amounts relative at circRNAs of production endogenous flanking sequences resulted in the plasmid information in Online Methods). The (detailed repeat or complementary 1-kb a with pairs); Alu complementary the include (which sequences flanking endogenous their 3 the 5 (retaining sequences flanking without circPAIP2 and circEIF3J to corresponding ( Alu We than constructed plasmids other with DNA sequences pairs sequence complementary flanking contained others four and pairs, complementary Alu flanking contained circRNAs 15 the of 4 of regions circSry of circularization the mediate might repeats inverted flanking that indication also was there and elements, Alu complementary contain to likely more were circRNAs forming Previous bioinformatic analysis showed that sequences flanking exons EIciRNAs nature structural & molecular biology molecular & structural nature Student’s two-tailed circEIF3J Name Table 1 circMIER1 circFUNDC1 circRSRC1 circPAIP2 UPR, theupstreamregionof junctions are shown. Exon 4-5(P) indicates that part of the known exon 5 sequence was involved in the circularization. Parental genesequencesinvolvedinhead-to-tailsplicingtoformjunctionsandthelineardistancesof circCDK11B circCLTC circMAN1A2-3 circBPTF circNAP1L4 circMAN1A2-2 circMAN1A2-1 circRBM33 circWDR60 circSSR1 a ′ ′ ) Schematics showing that genomic regions regions genomic that showing ) Schematics splicing site with the conserved GT); with with GT); conserved the with site splicing splicing site with the conserved AG and and AG conserved the with site splicing b – d , error bars, s.e.m. from triplicate triplicate from s.e.m. bars, , error d ) As in in ) As

Sequences related to circularization. circularization. to related Sequences The The top 15 circRNAs enriched in the pool RNA-sequencing from Pol II CLIP b

) Overexpression of circEIF3J and and circEIF3J of ) Overexpression can b P

be , measured in HEK293 nuclei. nuclei. HEK293 in , measured < 0.05; ** < 0.05;

overexpressed c t Parental gene test. ) As in in ) As CDK11B CLTC MAN1A2 BPTF NAP1L4 MAN1A2 MAN1A2 RBM33 WDR60 SSR1 MIER1 FUNDC1 RSRC1 PAIP2 EIF3J 30 P CDK11B b < 0.01 by < 0.01 , for HEK293 HEK293 for 3 1 . The genomic genomic The . gene.

with i. 2 Fig. UPR-exon2 Exon 2-30 Exon 2-6 Exon 23-29 Exon 2-14 Exon 2-5 Exon 2-4 Exon 3-5 Exon 2-4 Exon 2-3 Exon 6-9 Exon 4-5(P) Exon 2-3 Exon 2-3 Exon 3-4

Junction

their a ). ).

flanking

advance online publication online advance a b Relative RNA level Relative RNA level

1,500 2,000 2,500 3,000 1,000 Linear distanceofhead-to-tailjunction(nt) 270 bp sequences Exon 10 12 14 16 Vector control Vector control 500 20 40 60 80 1,550 bp 0 0 0 0 0 0 3 300 bp 300 bp circPAIP2 exon circEIF3J exon Complementary Alupai circPAIP2 exon-intron 1,270 bp circEIF3J exon-intron 1,000 bp 1 920 bp 1 UPR 35. Exon 2 3. Exon 3 ** ** Complementary repeat 0

circPAIP2 flanking 900 bp 8

circPAIP2 circEIF3J flanking circEIF3J HeLa 50. // 8. ** ** Exon 4 6 Exon Exon 5 64,072 41,533 40,140 30,550 27,979 18,464 12,646 120 bp 480 bp circPAIP2 1 kb 8,042 6,837 6,480 5,102 3,364 1,889 548.

33.6 circEIF3J 1 kb that flanking repeat sequences could facilitate circularization of the the of circularization reported facilitate could groups sequences repeat two flanking that work, this publishing of process the During RNA ** ** Exon 985 647 4 3 r 1 2,314. Exon 340 bp 123.5 1,190 bp ** 2 ** 32 5 8 s , 3 3 c and that the circRNA sequences could possess internal internal possess could sequences circRNA the that and Relative RNA level Relative RNA level Exons formingcircRNA 10,000 12,000 14,000 16,000 18,000 20,000 2,000 4,000 6,000 8,000 1,000 1,200

Vector control 200 400 600 800 Vector control CDK11B RBM33 PAIP2 EIF3J 0 0 circPAIP2 exon circEIF3J exon circPAIP2 exon-intron circEIF3J exon-intron 1 1 11. 77 ** circPAIP2 flanking ** circEIF3J flanking 9 circEIF3 circPAIP2 HEK293 ( for circPAIP2 ~123 and for circEIF3J of ~2,310 overexpression fold a produced repeats circPAIP2 in HeLa cells; 1-kb complementary n te icN sqecs ol pos could characteristics. circularization internal sequences sess circRNA the and RNA, the of circularization facilitate could plasmids, it appeared that sequences flanking 3a Fig. intron retaining ( also were circRNAs overexpressed the and of the corresponding circRNA in the nucleus, sion plasmids also resulted in higher amounts ( cells HEK293 in circRNA similar had for circPAIP2 ( amounts of ~35-fold for circEIF3Jrelative and lower ~3-fold the with although circRNAs, of production the in resulted also sites 5 cing the just with but sequence flanking without circPAIP2 and circEIF3J Fig. 2 Fig. s 6. ** 46 ** 5 1,759 bp 157 bp 400 bp circPAIP2 1 kb circEIF3J 1 kb 1,893 J 768 bp 79. Other complementarypair ** ** Exon notforcircularization Exon Exon b Exon 23 ). From the data from these artificial artificial these from data the From ). 5 14,698 1,031. ). DNA sequences corre sequences DNA ). ** 164 bp ** E 2 2 Exon 2 1 xon 3 500 bp d Fig. Fig. 2 Relative RNA level Relative RNA level 800 bp 1,000 1,200 1,400 Fig. Fig. 2 1,000 bp 100 200 300 400 500 600 Vector control Vector control 200 400 600 800 Fig. 2 Fig. Exon 0 0

circPAIP2 exon b - circEIF3J exon Exon 29 circPAIP2 exon-intron ). These constructs also overexpression effects effects overexpression circEIF3J exon-intron 1,048 bp d 1 1 3 and 539 bp c Nuclear circPAIP2 Exon Nuclear circEIF3 ). The overexpres The ). 3. 1. 1,014 bp Intron s e l c i t r a * * circPAIP2 flanking E 6 9 195 bp

circEIF3J flanking xon HEK293 Supplementary Supplementary 4 14. 2. ** * 3 ′ 4 n 3 and 1,109. 7

circPAIP2 1 kb s

44. circEIF3J 1 kb ponding to to ponding ** ** 0 4 BPTF SSR RSRC1 WDR60 J 1,107. 477. ** ** ′ 1 spli 6 9  ­ - -

© 2015 Nature America, Inc. All rights reserved. PAIP2 cells the of half than ( more in genes parental corresponding their of loci genomic the with colocalized each circPAIP2 and circEIF3J ( also had on no effect substantial the levels of Fig. 4a had no on effect the levels of or circEIF3J circPAIP2 ( EIF3J ( respectively of transcription the on effect no had siRNA with in nuclear run-on experiments, knockdown of ( respectively levels, cEIF3J and circPAIP2 resulted in lower We of experiments. for nuclei cir found cell that run-on knockdown extracted then and siRNAs corresponding their with circPAIP2 and Wenuclear experiments. run-on performed down knocked circEIF3J resulted from a in decrease the of transcription the parental genes, we 3d Fig. ( genes neighboring the on effect no had circPAIP2 and circEIF3J of knockdown that found and 5 the Supplementary Fig. 3b and HEK293 cells, respectively ( HeLa in genes parental the the of in levels mRNA decrease a in resulted EIciRNA ing (ASOs) oligonucleotides antisense H–based RNase or (siRNAs) RNAs fering inter short with circPAIP2 and circEIF3J of knocking down their expression. Knockdown by EIciRNAs of roles evaluate to Westarted EIciRNAs in genes parental their of expression the regulate could EIciRNAs whether tigate to inves plasmids overexpression these used characteristics circularization Student’s two-tailed ** transfections. triplicate from s.e.m. bars, error figure, Throughout and examined, cross- were genes two The knockdown. circRNA after gene parental corresponding the of transcription in decrease a specific ( in specified (sequences junction EIciRNA the targets ASO the circPAIP2, For intron. a downstream targets ASO control the and EIciRNA, the in sequences intron targets ASO the circEIF3J, For ASOs. with circPAIP2 or circEIF3J of knockdown after genes parental the of levels mRNA in ( black. in shown are siRNA control corresponding the in sequences mismatched 3 5 corresponding the show histograms the below sequences purple and blue The sequences. junction at circPAIP2 and circEIF3J targeted siRNAs The siRNA. with circPAIP2 or circEIF3J of knockdown after genes parental ( effects. 3 Figure s e l c i t r a  ( EIciRNAs two the with colocalization no demonstrated c Fig. 4a Fig. Supplementary Fig. 4d Fig. Supplementary

′ ) Nuclear run-on experiments showing showing experiments run-on ) Nuclear exon sequences forming the junction; junction; the forming sequences exon ′ neighboring genes of genes neighboring or or at the 5 – ). To further examine whether the decrease in mRNA levels levels mRNA in decrease the whether examine Tofurther ).

, c a b EIciRNAs have have EIciRNAs PAIP2 ). ). Overexpression of circEIF3J and circPAIP2 from plasmids ) Decrease in mRNA levels of the the of levels mRNA in ) Decrease ). This finding is in contrast with genes that flank flank that genes with contrast in is finding This ).

regulate Supplementary Fig. 3f Fig. Supplementary ′ GAPDH and 3 mRNA with short hairpin RNA (shRNA) or siRNA siRNA or (shRNA) RNA hairpin short with mRNA t

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EIF3J PAIP2 circEIF3J siRNA-control circEIF3J siRNA circEIF3J junction a c b and and EIF3J Relative RNA level Exon Relative RNA level Supplementary Supplementary Supplementary Supplementary Relative mRNA level (normalized with GAPDH) GAPDH (normalized with GAPDH) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.2 0.4 0.6 0.8 1.0 1.2 3 1.2 0.2 0.4 0.6 0.8 1.0 PAIP2 ). ). In addition, PAIP2 transcription 0 0 0 EIF3J circEIF3J and and circEIF3J Control Intron Scrambled advance online publication online advance Fig. 4 Fig.

EIF3J ASO siRNA EIF3J circEIF3J siRNA Scrambled siRNA mRNA mRNA mRNA EIF3J circEIF3J ** 3 , , which PAIP2 circEIF3J siRNA circEIF3J siRNA-contro 5 ′ circEIF3J E ** 0.006 xon mRNA or 3 3 c ′ ′ circEIF3J ). ).

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PAIP2 ** ** U1A U1A and U1C proteins and U1 nuclear small RNA (snRNA; or circEIF3J circPAIP2with either not only yielded Pol II but the also in pulldowns these of specificity ( EIciRNAs specific with experiments) (ChIRP) purification RNA by isolation chromatin (in coprecipitated DNA genomic examined also we chroma tin, and EIciRNAs between interactions potential investigate to Furthermore, EIciRNAs. two the with proteins coprecipitating the RNAs and analyzed we sequences, EIciRNA specific to mentary comple oligonucleotides biotin-labeled with assays Using pulldown EIciRNAs genes. parental their the on focus to genes. chose we parental study, their this In than other loci on not EIciRNAs these was of effects nucleus potential the indicating thus in loci, gene EIciRNAs parental their to two confined these in of genes parental localization the their of expression the may circPAIP2 and regulate circEIF3J that suggest data these Collectively, down with the circRNAs, but it appears that the association between between association the that but it appears circRNAs, the with down and circEIF3J ASO siRNA-control circEIF3J upeetr Fg 5b Fig. Supplementary Exon 4 l GAPDH circEIF3J controlASO Exon 3

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U ). U2 snRNA could also be pulled pulled be also could snRNA U2 ). PAIP2 1. 1. 0. 0. 0. 0. circPAIP2 siRNA-1 Scrambled siRNA mRNA Supplementary Fig. 5a Fig. Supplementary 2 5

** Scrambled 0 2 2 4 6 8 0 1 circPAIP2 ′ mRNA siRNA-1 circPAIP2 siRNA-1 circPAIP2 siRNA1-contro circPAIP2 siRNA-2 circPAIP2 siRNA2-contro E snRNP ** xon ** ASO 3 3 ′ ′ 3 cis EIF3J circPAIP2 ** circPAIP2 siRNA1-

3 3 control and ′ ′ effects of EIciRNAs on on EIciRNAs of effects ASO

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circPAIP2 ASO promoters ** cis 5 5 circPAIP2 Intron ′ ′ ). Pulldown Pulldown ). . However, However, . circPAIP2 siRNA2- control Fig. Fig. 5b 2 5 5 Exon ′ ′ trans 3 3 ′ , c - -

© 2015 Nature America, Inc. All rights reserved. without colocalization. The circRNA FISH signal was detected with a junction probe in HEK293. Scale bars, 10 10 bars, Scale HEK293. in probe a junction with detected was signal FISH circRNA The colocalization. without 5 of loci genomic the with colocalization circRNA no showing panel) image ( images FISH double ( circPAIP2). for cells 100 and ( circPAIP2 for ~56% and circEIF3J for ~54.4% are percentages the and cells, the of half than more in circRNA the with A + B) (categories colocalized are loci gene parental Both graph. bar in shown is categories four the for analysis Statistical A. category in shown Images circRNA. with colocalized is locus neither D, circRNA; with colocalized not, is one other the and is, locus gene one C, circRNA; with, colocalized and of, edge the at localized are loci gene both B, signal; circRNA the inside localized are loci gene both A, loci: gene parental and circRNA of colocalization the in categories ( stain. 4 DAPI, loci. gene parental corresponding the and probe) junction a with (detected circRNAs of colocalization and circEIF3J ( loci. gene neighboring or parental its 4 Figure green; green; Y/R, ratio of yellow to red. Scale bars, 10 of the of with majority a the colocalization (detected with showing probe) circRNAs U1 junction snRNA (Y/R in ratio) Y/G, cells. HEK293 ratio of to yellow * experiments. triplicate from s.e.m. For comparison. for shown are U2AF65 and U2AF35 for Results U1C. or U1A to antibodies with assay ChIP by promoters gene parental ( Methods). Online in described (as detected not ND, comparison. for shown are Lsm10 and U2AF65 U2AF35, for Results U1C. or U1A to antibody an with IP RNA in EIciRNAs ( oligonucleotide. Oligo, circRNAs. indicated the against ChIRP in ( marker. M, controls. negative are rRNA 5S and snRNA U7 circRNA. with snRNA U1 of in shown are blots of images Uncropped controls. negative are HDAC2 and snRNP) U7 of component protein (a Lsm10 circRNA. with together U1C and U1A II, Pol of pulldown showing presented in the experimental setup for the data gene promoters. ( parental and snRNP U1 II, Pol 5 Figure We repeated these experiments with another set of biotin-labeled biotin-labeled of olig set another with experiments these repeated We ( indirect be may circRNA and snRNA U2 nature structural & molecular biology molecular & structural nature a d ′ ) Representative FISH images ( images FISH ) Representative gene gene ) Pulldown of the parental gene promoters promoters gene parental the of ) Pulldown Supplementary Data Set 1 Set Data Supplementary o a isolatio Protei nucleotides complementary to exonic sequences in EIciRNA EIciRNA in sequences exonic to complementary nucleotides Pol II ( b b ) MATR3 ) FISH images demonstrating four four demonstrating images ) FISH

n for circEIF3JorcircPAIP Pulldow n Double FISH for circRNA and and circRNA for FISH Double EIciRNAs interact with with interact EIciRNAs Biotin-labeled probes b – n n isolatio d and 3 and = 16 for circPAIP2) showing showing circPAIP2) for = 16 RNA . ( Pol II ( a c ) Flow chart of ′ ) b Cross-lin ,6-diamidino-2-phenylindole ,6-diamidino-2-phenylindole ) Western blots n n n ′ gene gene = 90 cells for circEIF3J circEIF3J for cells = 90 = 40 cells for each each for cells = 40 e GAPDH k ) Pulldown of ) Pulldown isolatio ChIRP 2 DNA a ( d belong to the the to belong PROB1 ) . . ( n c c

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© 2015 Nature America, Inc. All rights reserved. Pol II might interact with each other at promoter regions of the paren and snRNP U1 EIciRNAs, that suggesting genes, parental the of site start transcriptional the of upstream bp ~300 region a occupy U1C) results ( as such genes of promoters the to not as such genes, some of promoters the to binds ( ment gene ( of for neighboring promoter the the or U2AF65 U2AF35 U1C, U1A, chromatin by demonstrated immun as genes, parental corresponding regions of promoter the with interacted U2AF35 or U2AF65 not but result in substantial enrichment in EIciRNAs ( (U2AF65 and U2AF35) for U2 recognition of the 3 factors auxiliary or (snRNP) ribonucleoprotein nuclear small U7 of component protein (a Lsm10 with pulldown EIciRNAs; of amount a substantial coprecipitated or U1A proteins the U1C with pulldown Error bars, s.e.m. from triplicate experiments. circRNA, U1 snRNA and FISH parental-gene-loci images is shown in probe) and U1 snRNA in ~65.4% of cells for Figure 8a and U1C to the promoters of parental corresponding genes upon circRNA knockdown via siRNA. For genes ( reduced Pol II binding to the parental gene promoters upon circRNA knockdown via siRNA. Data are also shown for promoters of neighboring corresponding pEIF3J(B), the B site shown in AMO. with snRNA U1 of blocking the without or with circRNA corresponding ( controls. are AMOs U2 and Scrambled shown. also are RNA 7SK and snRNA U2 snRNA, U1 for Data circRNAs. with II Pol of binding of ( control. a negative is EIciRNA), to rise give not does (but gene intron-containing of transcription decreased ( controls. are AMOs U2 and Scrambled qPCR. with examined were gene parental the of mRNA and circRNA of Levels AMO. with snRNA U1 of ( 6 Figure s e l c i t r a  that RNA for FISH EIciRNA and Dual U1 snRNA revealed genes. tal a d d b a

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© 2015 Nature America, Inc. All rights reserved. and EIciRNA examined with Pol II CLIP also decreased upon AMO AMO upon decreased also CLIP Pol II with examined EIciRNA and antisense with ( oligonucleotides biotin-labeled pulldown EIciRNA by examined as snRNA EIciRNA, U1 and between interaction decreased AMO with site this ing at the 5 EIciRNAs, potentially two the of each in site snRNA–binding U1 one only be might there complementarity, sequence of basis the On EIciRNAs. We the where U1 snRNA next examined site binding in is the located U genes. of parental expression on the of EIciRNA effects the mediates Supplementary Fig. 7b ( cells the of half than more in loci gene parental around concentrated are snRNA U1 and EIciRNA that showed images FISH ( promoter gene the at binding II Pol on 8c Fig. upon the knockdown of the corresponding circRNA ( decreased also was genes parental the of bodies gene the to binding ( promoters gene nature structural & molecular biology molecular & structural nature blocking of the U1-binding site ( * experiments. triplicate from s.e.m. bars, error figure, Throughout AMO. EIciRNA corresponding the with treatment after promoter gene parental the and ( cross-examined. were genes two The AMO. EIciRNA with treatment ( AMO. EIciRNA with treatment after ( cross-examined. were genes two the and control, AMO. EIciRNA with treatment after gene parental corresponding the of transcription decreased showing experiments run-on ( altered. significantly not was II Pol to binding RNA 7SK and snRNA U2 snRNA, U1 of amount total The effect. specific sequence the show to cross-examined were EIciRNAs two The AMO. EIciRNA with treatment after EIciRNA corresponding the and II Pol between association decreased showing experiments ( control. a negative is sequences intron the targeting AMO AMO). (EIciRNA EIciRNA the in snRNA U1 of 5 (the site binding the targeting AMO with treatment after snRNA U1 and EIciRNA between association decreased showing experiments pulldown ( EIciRNAs. of effect the for essential is 7 Figure control AM circPAIP2 AM circPAIP2 control AM circEIF3J AM circEIF3J a c P 1

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0. 0. 0. 0. 1. 1. 0. 0. 0. 0. 1. 1. 2 4 6 8 0 2 2 4 6 8 0 2 0 0 interact with the Pol II transcription complex at the promoters of of promoters the at complex transcription II Pol the with interact EIciRNA, and then the EIciRNA–U1 snRNP complexes might further and snRNA U1 between RNA-RNAinteraction through snRNP U1 as such factors hold might EIciRNAs circPAIP2). and circEIF3J ple, Here we a identified class special of circRNAs as EIciRNAs (for exam DISCUSSION is EIciRNA in site EIciRNAs. of effect U1-binding transcription-enhancing the for essential the between via EIciRNA interaction and RNA-RNA snRNA U1 specific the Thus EIciRNAs. by enhancement transcriptional of process whole the as well as aspect every for critical is EIciRNA to snRNA U1 of binding the that strate ( blockages AMO these by attenuated gene promoter, ChIRP,with parental examined were substantially all ChIP, the of with EIciRNA with promoter, examined and interaction ( decreased also Pol and were II promoter gene parental the between interactions the ( experiments run-on nuclear in EIciRNA gene parental corresponding the of the transcription in the snRNA decreased U1 of site binding the of circEIF3J AM circEIF3J controlAM circPAIP2 AM circPAIP2 controlAM pPAIP2(D) pEIF3J(C) b Relative RNA enrichment 0. 0. 0. 0. 1. 1. 1. 1. ** ** 2 4 6 8 0 2 4 6 circEIF3 0 pCTDSPL * pMATR3

circEIF3J AM circEIF3J controlAM O J O circPAIP2 Fig. Fig. 7 O O 2 Fig. 7 Fig.

U1 c O e ). Consistently with the decreased transcription, transcription, with the decreased ). Consistently Relative DNA enrichment Relative DNA enrichment d 0. 0. 0. 0. 1. 1. 1. 0. 0. 0. 0. 1. 1. 1. ). Binding of U1 snRNP to the parental gene gene parental the to snRNP U1 of Binding ). 2 4 6 8 0 2 4 2 4 6 8 0 2 4 0 0 U2 O circEIF3J AM circEIF3J controlAM circPAIP2 AM circPAIP2 controlAM pPAIP2(D) pEIF3J(C)

7S ** ** K pPAIP2(D) O pEIF3J(C) O Relative RNA enrichment 0. 0. 0. 0. 1. 1. 1. 1. Fig. 7e Fig. 2 4 6 8 0 2 4 6 circEIF3J 0 O O GAPDH circPAIP2 AM circPAIP2 controlAM circPAIP2 , f f ). These data demon data These ). Relative DNA enrichment Relative DNA enrichment ** 0. 0. 0. 0. 1. 1. 0. 0. 0. 0. 1. 1. is a negative a negative is s e l c i t r a circEIF3J AM circEIF3J controlAM circPAIP2 AM circPAIP2 controlAM 2 4 6 8 0 2 2 4 6 8 0 2 0 0 O pPAIP2(D) U1 pEIF3J(C) ** ** U2 O O O

7S

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© 2015 Nature America, Inc. All rights reserved. flanking repeat sequences and biogenesis of circRNAs of biogenesis and sequences repeat flanking ( work EIciRNAs. of mechanisms and roles regulatory potential other exclude not do of mechanism ncRNAslying the functional under themes central the of one be might EIciRNA and snRNA U1 Specific RNA-RNA interaction between ncRNAs such as that between functions in the cytoplasm the in functions also is regulatory It in involved be may circRNAs EIciRNAs. exon-only that and fascinating circRNAs ‘noncoding’ of exonic of types two transcripts least at circular of formation the to sequences, contribute ‘protein-coding’ as viewed classically been have which sequences, exonic certain Also, cells. in animal circRNA populations intronic or exonic sequences either with exclusively formed circRNAs with together study, this in EIciRNAs of identification The interaction. RNA-RNA via works that mechanism fine-tuning gene-expression a into insight new snRNP, U1 provide we with interaction via genes high. be to need sarily for EIciRNAs, circRNAs of not the does abundance neces individual circRNAs most for tion func a regarding biological and raised have doubts been abundance, introns cells retained mammalian have in might circRNAs of thousands the of ~20% microRNA analysis, as act could them) sponges of several just (maybe circRNAs regulation. gene and sion Fig. 7 ( cells tions among individual and distribu amounts in their of heterogeneity degree some showed also detail in characterized we that EIciRNAs two The mRNA. and the parental genes transcriptionally to increase levels of both circRNA However, of EIciRNAs expression the may once generated, modulate splicing during RNA precursor linear single the from generated is the tion linear mRNAbetween and the circRNA regarding which one competi of relationship a is there that possible It is specificity. gene circRNAs of seems to be regulated by some biogenesis mechanisms with cell and/or sequences, nucleotide the Beyond circCLTC. express cells HeLa only although cells, HEK293 and HeLa both in expressed circCLTC for observations ( our to similar specificity cell or tissue circRNA any described previously not have researchers knowledge, To at of spot as are the our transcription. long as generated tion, they scriptionally or post-transcriptionally is not an issue for their debate is a still or topic ated under post-transcriptionally cotranscriptionally by us ( as noticed biogenesis, to circRNA contribute also sequences internal premature preventing pol events, transcriptional early stimulating such as roles, other has snRNP U1 that shown have evidence of lines of U1 snRNA is splicing function conventional The tional level by ncRNAs is a fundamental aspect of gene expression at Regulation the transcrip feedback. tion, positive hence generating of EIciRNA from the gene would further promote the gene’s transcrip ulate that once the transcription of a gene is turned on, the generation parental genes to enhance gene expression (model in s e l c i t r a  transcriptional for retained efficiently be to appear study this in Supplementary Fig. 1f Fig. Supplementary

By showing that EIciRNAs promote the transcription of parental parental of transcription the promote EIciRNAs that showing By limited very only that raised been has argument an Recently, Our of research. hotspot a recent of been has circRNAs Biogenesis y dnlto ad eemnn pooe directionality promoter determining and adenylation ), ), and this may indicate certain dynamic features of gene expres Fig. 2b Fig. 33 Fig. Fig. 2 , 4 9 4 . It has also been noted that, according to bioinformatic bioinformatic to according that, noted been also has It . 27– . In our model, whether EIciRNAs are generated cotran generated are EIciRNAs whether model, our In . 2 ) and several publications have shown a link between between link a shown have publications several and ) 29 – d , 4 ) and also ) by and others also 5 , suggests that there are at least three distinct distinct three least at are there that suggests , 2 ). The mRNA of the CLTC-encoding gene is gene CLTC-encoding mRNA of the The ). 9 . For the the For . 27 2 Figs. 4 Figs. 9 , . The majority of circRNAs have low low have circRNAs of majority The . 2 8 , whereas the EIciRNAs identified identified EIciRNAs the whereas , cis 3 , 3 5g effect, such as we present here here present we as such effect, . Whether circRNAs are . gener Whether and 4 3 6 . . However, our data also g , , and 3 Fig. Fig. 7 Supplementary Supplementary , but additional , but additional 8 30 ). ). We spec , advance online publication online advance 32 cis , 10 3 3 func , . The The . 38– 35 , 4 4 3 2 4 6 ------­ . . .

5. 4. Methods and any associated references are available in the the the of in version available are references associated any and Methods M circRNAs. of roles physiological the explore rigorously to performed be need will studies further diverse, therefore and rather be may circRNAs of mechanisms related and tions func the that speculate we Furthermore, nucleus. the in regulation 3. 2. 1. reprints/index.htm at online available is information permissions and Reprints The authors declare no competing interests.financial analysis. All authors the resultsdiscussed and made comments on the manuscript. and (or) data analysis. X.W., P.Z., Z.C., Q.W. and Y.Z. performed bioinformatic X.W., G.Z., B.Y., W.H., L.D., Y.J., P.X. and H.L. performed some of the experiments performed most of the experiments and analyzed most of the data. L.C., M.L., G.S. wrote the manuscript with the assistance of Z.L. and C.H. Z.L., C.H. and C.B. G.S. conceived this project, their experiments designed execution. and supervised FoundationScience of China, 11175068). Foundation of China, 81372215 and 31301069) and Y.J. (the National Natural Universities of Z.L. China, WK2070000034), (the National Natural Science and KJZD-EW-L01-2; and the Fundamental Research Funds for the Central 91232702 and 31471225; the AcademyChinese of 1731112304041 Sciences, and the 2015CB943000; National Natural FoundationScience of China, 81171074, by grants to G.S. (the National Basic Research Program of China, 2011CBA01103 Shan laboratory for discussions and technical support. This work was supported S. Guang, X. Song, Q. Liu, Y. Mei and M. Wu for discussions and members of the The authors thank S. Altman, Y. Shi, R. Chen, E. Wang, W.W. Walthall, P. Jin, version of the pape Note: Any Supplementary Information code and Source Data files are available accession in the under database GSE6444 Omnibus Expression Gene the codes. Accession C AU A genes to enhance gene expression. gene enhance to genes parental of promoters the at complex transcription II Pol the with interact further might complexes snRNP EIciRNA–U1 the then and EIciRNA, and snRNA U1 between interaction RNA-RNA specific through snRNP U1 as such factors hold might EIciRNAs genes. parental of expression 8 Figure c O

ethods kno T MP i, H. Liu, unconventional RNAs: noncoding from Signals G. Shan, & L. Chen, J., Wu, S., Hu, Ulitsky, I. & Bartel, D.P. lincRNAs: genomics, evolution, and mechanisms. small for roles nucleus: the in interference RNA R.A. Martienssen, & S.E. Castel, J.T.Lee, RNAs. noncoding long by regulation Epigenetic physiology of physiology (2012). roles for conventional pol III transcripts. (2013). 26–46 (2013). beyond. and epigenetics transcription, in RNAs (2012). U1 SNP H O ET wl R R C

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© 2015 Nature America, Inc. All rights reserved. RN00196 sPI22 TC00137) ih eaie shRNA negative with TRCN0000153174) shPAIP2-2, TRCN0000219946; TRCN0000062016; shEIF3J-2, TRCN0000062017) and confirmation. The shRNA plasmids for knockdown of biotin-oligo are listed in primers for all plasmid construction, probe preparation,for Oligos recombinant siRNA,methods. with or ASO,ligation and digestion AMO,tion-enzyme and Plasmids and plasmid construction. of northern blots can be found in ImageQuantBiomolecularLAS4000 Imager Healthcare).(GE imagesOriginal ing to the manual (Roche, DIG Northern Starter Kit). Images were taken with an 0.1× SSC and 0.1% SDS at 62 °C for 30 min, and detection was wasperformed performedat 62 °C accordovernight. Membranes were stringently washed twice in onto Hybond-N circEIF3J_exon/intron or circPAIP2_exon/intron plasmids. RNA wasin MOPS buffer.transferred In the O.E. lanes, total RNA was from overexpression with the loaded on a 2% agarose gel containing 1% formaldehyde and were run for 1.5 h digestionR andRiboRuler High RangeRNA (ThermowereScientific) Ladder RNase without or Total forwith manual.RNA the templatesto accordingtranscription T7 as products PCR corresponding the with (Roche) Kit Starter Northern blot. detect the breakpoints flanked by GU/AG splice sites. anchoruniqueanchortheextended alignmentsfind wepositions,to then and reads, we extracted 20-mers from both ends and aligned them independently to reads that aligned contiguously and full length to the genome;colleagues from the remaining used to predict circRNAs according to the approach establishedwere readsremaining the and first, byoutfiltered were nt Rajewsky 50 than shorter and Reads was downloaded from the UCSC genome browser ( library and the sample (Pol II-CLIP) library, respectively. ReferenceIn total,genome we obtainedhg19 8,975,835 and 5,088,901 reads from the control (IgG-CLIP) and were applied to an Illumina GAIIx system for 80-nt single-end sequencing. CLIP RNA samples were prepared according to the manufacturer’s instructions RNA sequencing and bioinformatic analysis. antibodies used, validation is provided on the manufacturers’ websites. AGO2 from Sigma (cat. no. SAB4200085) was used. For these and all the other immunoprecipitation.to CLIP,antibodyAGO2II monoclonal PolFor awith DNase I (Promega) to the cell suspension after sonication and then followed this DNA was necessary for the association between Pol II and circRNAs, we added without performed withPol ForII. later verification ofenrichment ofcircRNAs, assaysCLIP were tion, and it presumably would reduce the association of newly synthesized CLIP.RNAsfor used was ab5095)no. cat. ing, and anti-RNA polymerase II CTD phospho-S2 antibody,HeLa cellsChIP gradewere (Abcam,pretreated with 20 (pH 4.2) extraction to obtain RNA. To prepare CLIP RNA for deep sequencing, with 30 (100 mM Tris, pH 7.8, 10 mM EDTA, and 1% SDS) and werethe antibody–Proteinreverse cross-linked G bead complexes were resuspended in 50 one-fifth volume of solution after the last wash was saved for western blots. antibody–ProteinThen G bead complexes were washedThe °C. five4 at h times3 least atfor with CLIP perform to lysisadded was suspensionFlowFast buffer, and 4 G–SepharoseProtein then and °C, 4 at h 1 forantigencouple to added was 4 °C (Input). Anti–Pol II antibody (Abcam, cat. no. ab5095) or IgG (as control) at h 1 forAmersham) (GE suspensionFlow Fast 4 G–Sepharoseprotein with 12,000at centrifuged wassuspension cell Bioruptorthe (Diagenode),a with min 15 for 1.5 mM DTT, and 1× protease-inhibitor cocktail (Sangon). Cells were sonicated and 0.5% Triton-X 100), 100 units/ml RNasin Plus RNase Inhibitor (Promega), EDTAmM NaCl,30 mM 200 7.4, pH HEPES, mM (10buffer lysisice-cold in irradiated in a UV cross-linker (254 nm, 400 mJ/cm previously described with some modifications assay. (CLIP) immunoprecipitation Cross-linking ONLINE nature structural & molecular biology molecular & structural nature (Sigma). MISSIONshRNA Library the fromobtained controlwere(SHC002) µ g of proteinase K at 65 °C for 1 h; this was followed with phenol/chloroform g for 5 min at 4 °C, and the supernatantpreclearedandthe andcollected °C, was 4at min for5 2

7 METHODS . In brief, we aligned reads to the referencethe genometoreadsdiscarded and aligned webrief,In . Digoxin-labeled RNA probes were prepared with DIG Northern + membranes (GE Healthcare) by capillary transfer. Hybridization α -amanitin. To analyze whether the presence of genomic of presence the whether Toanalyze-amanitin. Supplementary Table 4 µ Supplementary Data Set 1 g/ml α All plasmids were constructed with restric -amanitin was used to inhibit transcripinhibit to used was-amanitin α -amanitin (Sigma) for 6 h before harvest For high-throughput sequencing, 4 . All plasmids were sequenced for 6 . Briefly, the cultured cells were 2 http://genome , 1 min) and then harvested CLIP was carried out as out carried was CLIP PAIP2 EIF3J mRNA (shPAIP2-1, mRNA (shEIF3J-1, . µ l elution buffer .ucsc.edu / ) 4 7 - - - - .

were labeled with Alexa Fluor546, Fluor488, or Fluor647 with the correspondingthe insertion in ULYSISthe T vector as a template for transcription,Nucleic and with Scientific), (Thermo TranscriptionYieldKit High TranscriptAidT7 the Fluorescence real-time PCR using the synthesized cDNA. each circRNA was calculated on the basis of cell numbers and the Ct value from 10 × 1.0 numbercopy inHeLa cells was calculated circRNAasfollows. Total PCR. RNA was extractedfrom real-time by curve standard a plot to used was dsDNA purified then andcDNA, withcircPAIP2 amplifiedor were circEIFP3J to ing Quantification of circRNA copy number per cell. plasmids can be obtained upon request. pEGFP-C1 for these overexpression constructs. Further5 information1-kb the about of these sequences ­complementary circRNA,formingsequencesthe reverse-and the kb), genomic(~1 sequences plasmidsinsertionswith corresponding sequencestoincluding 5 the the plus circRNA5 the forming sequences to corresponding plasmids insertions are with circPAPI2_flanking and circEIF3J_flanking insertion). the in the 5 circRNA(with to the formingexon/intron sequences corresponding insertions with plasmids are circPAPI2_exon/intron 5 (with are plasmids with inserts corresponding to the two exons forming the circRNA For the overexpression of circRNAs ( (AMOs), including U1 AMO, U2 AMO, and scrambled intoAMO, phosphorothioatewere synthesized oligonucleotidesat ribonucleotides, were synthesized (RiboBio). All bases of ASOs were converted modified by changing the five nucleotides at the 5 protocol. 2- Oligofectaminestandard(Invitrogen)or the 2000 accordingLipofectamineto performed with the transfected cells. Transfection of siRNA was conducted with DNAmidwith sure that the transfection efficiency was about the same, real-time PCR for plas Lipofectamine(Invitrogen)2000 according supplier’stheto protocol. To make CO 5% and °C 37at FBS plus10% and HeLa cells were maintained under standard culture conditions with DMEM culturetransfection and Cell ofplasmids,siRNA, ASO, AMO.and ment was used as background subtraction. ImageJ. The area (same size as the band below) just above the band of measure Quantification of band intensity from ImageJ.northernby merged blotting also werewas channels also color performedand software, image-acquisitionwith Image processing and quantification. software. laser confocal microscope system (Andor Technology), with the Andor IQ 10.1 XD Revolution Andor an on mounted camera CCD EM DV897K iXonEM+ microscopy. Confocal antibody (Life Technologies, ab150074). for 4 h; this was followed with incubation with Alexa Fluor555–labeledwereincubated antibodywith against secondary Immunofluorescence staining. if not specified. regular FISH protocol. FISH signal for circRNA was detected withRNase R atjunction 37 °C for 30 min and then fixed again beforeprobe being subjected to the ForFISH withRNase digestion,R cellswerefixed washedPBS,treatedin with hybridizationthen sameconditionsperformedthewaswith foras RNA FISH. genomicwithULYSIS awith andlabeledPCRthen Nucleic Acid LabelingKit; ForDNA-RNAmin. 10 for °C DNApreparedFISH,doublewereprobes45 at (Life Technologies, final concentration 30 ng/ 80 °C for 10 min at anddenatured thenwere incubatedprobes RNA atand 42 intensity.Cells °C fluorescence for the 15–17 amplify h with human Cot-1 DNA Acid Labeling Kit (Invitrogen), which added a Fluor on Gevery of the probe to ′ and 3 and ′ -AG and 3 and-AG ′ 6 flanking genomic sequences. circEIF3J_1 kb and circPAPI2_1andcircEIF3J_1kb genomicsequences. flanking arekb cells, and cDNA was then synthesized. The copy number per cell of cell per numbercopy The synthesized. then cDNAwas and cells, O -methyl RNA/DNA antisense oligonucleotides (ASOs), which were in situ in GAPDH ′ -GT included in the insertion). circEIF3J_exon/introninsertion). and the in included -GT hybridization (FISH).hybridization loecne inl wr gtee wt a Andor an with gathered were signals Fluorescence genomicendogenousDNAcontrolanloading as was For 2 Fig. . Plasmid transfection was conductedtransfectionPlasmidwithwas . β 3 -actin immunostaining, slides after FISH β Cell images were processed with ImageJ 1 . Antisense Morpholino oligonucleotides -actin (Abcam,-actin ab8227,dilution)1:100 2 ), circEIF3J_exon and circPAPI2_exon ′ flanking sequence. The vector was vector The sequence. flanking µ RNA probes were transcribed by transcribed wereprobes RNA l). Slides were washed with 2× SSC ′ and 3 DNA fragments correspond ′ AG and 3 and AG ′ doi: ends into 2 10.1038/nsmb.2959 ′ GT included GT ′ - O ′ HEK293 flanking -methyl - - -

© 2015 Nature America, Inc. All rights reserved. regions with the corresponding PCR primer pairs. quantitative real-time PCR (qPCR) for the detection of enriched genomic DNA extracted with phenol/chloroform/isoamyl alcohol. Eluted DNA was subjected65 °C. to Samples were digested with proteinase K for 1 h at 45 in 1% (w/v)°C, SDS and 50 mMand NaHCO the DNA was eluted werecomplexes immune The SC-48804). no.Biotechnology, cat. Cruz (Santa U2AF65 and SC-19961), no. Biotechnology,cat. Cruz SC-101549), (Santa U2AF35 no. cat. Biotechnology, Cruz (Santa U1C SC-101149), no. cat. PolII(Thermo Scientific, cat. no. 01671887), U1A(Santa Biotechnology, Cruz chromatinThe solutionprecleared,was immunoprecipitated antibodytowith 16.7mMTris-HCl, pH8.1, and 167mMNaCl) containing protease inhibitors. ChIP dilution buffer (0.01% (w/v) SDS, 1.1% (v/v) in Triton1:10 diluted was remainingX-100, The input.1.2 as mM saved EDTA,was supernatant the of ple Bioruptor(Diagenode) toobtain up to500-bp DNA fragments. 100-A were incubated for 10 min on ice. Cell extracts were sonicated for 15 min with a Tris-HCl, pH 8.1) containing complete protease inhibitor cocktail (Roche) and resuspendedof ml SDS1in lysis buffer (1%(w/v)SDS, EDTA,mM10 mM50 0.125glycineM for min,and5 cellsthe were pelletedat 800 with quenched then temperature.was roomCross-linking at min 10 forhyde previously,modificationswith Chromatinimmunoprecipitation (ChIP). found in Biomolecular Imager (GE Healthcare). Original images of western blots can be Signaling (Cell anti-HDAC2 Technologies, and cat. no. 5113). Images were takenwith an ImageQuant ab180128) LAS4000 no. cat. (Abcam, anti-Lsm10 cat. no. SC-19961), anti-U2AF65 (Santa Cruz Biotechnology, cat. no. SC-48804), Biotechnology, Cruz (Santaanti-U2AF35Biotechnology, SC-101549), no. cat. Cruz (Santa anti-U1C SC-101149), no. cat.Biotechnology, Cruz (Santa U1A RNA polymerase II CTD phospho-S2 antibody (Abcam, cat.anti- no. ab5095), anti-blots: western in used were antibodies following The Healthcare). (GE protocol blotting western ECL the to according processed were Membranes rated on SDS-PAGE gels and then transferred to PVDF membranes (Millipore). Westernblot. (Abcam, cat. no. ab1893) and converted to cDNA for real-time PCR assay. treatment. Nascent transcripts were immunoprecipitated by anti-BrdU antibody themanufacturer’s instructions, and DNA was eliminated by DNase I(Takara) Technologies) per (Life reagent TRIzol by isolated was RNA The (Promega). inhibitor RNase of presence the in min 5 for °C 28 at incubated was nuclei) ATP,mM CTP, crude (10 GTP, mixture the BrUTP, run-on and nuclear The centrifugation. The nuclear run-on protocol was modified from Guang 0.5% NP-40). The crude nuclei were then prepared by sucrose density gradient pended in lysis buffer (150 mM KCl, 4 mM MgOAc, 10Tris-HCl, mM pH 7.4) Tris-HCl,and were pelleted by centrifugation. Then pelletspH were resus7.4, and vested in ice-cold hypotonic solution (150 mM KCl, 4 mM MgOAc, and 10 mM Nuclear run-on assay. AMO against EIciRNAs and the corresponding controls, 7.5 The final concentrations were: ASO, 5 electroporation was performed, and this was followed with cell harvest 12 h later.ASO transfection was performed twice: 36 h after the first experimentstransfection, 8 h theafter secondAMO transfection. To maximize the knockdown minimize side efficiency,effects of AMO, cells were harvested for analysis or downstream the Nucleofector system (Lonza) according to the manufacturer’sGene instructions.Tools. To ASO and AMO treatments were performed with electroporation with doi: 10.1038/nsmb.2959 Supplementary Data Set 1 For westernblots,lysates cellmixtureswhole andIP were sepa For nuclei isolation, cells were rinsed with PBS and har 4 9 . Cells were fixed on plates with 1% formaldeplatesonwere1% fixedwith Cells . . 3 µ , and cross-links were reversed for 6 h at M; U1 AMO and U2 AMO, 75 ChIP was carried out as described described outascarried was ChIP g µ . Cell pellets werepellets Cell . M. µ µ et al. M; and l saml 4 8 - - - - - .

then subjected to RNA elution, DNA elution, or protein elution. wash buffer (2× SSC, 0.5% SDS, and 0.1 mM DTT and PMSFwith Dynabeads volumeof the (fresh)).40× with Technologies)times five washed and Beads were (Life magnets by captured were Beads °C. 37 at min 30 for rotated then was pmolbiotin-DNAof100 per added mix waswhole oligos, theand Dynabeads nuclearagaininbufferbeforelysisresuspended. being 100 temperature,roomattimes h threewashedfor1 thenBSA mg/mlRNA 1 and three times in nuclear lysis buffer, which was blocked with 500 ng/ at 37 °C for 4 h. M-280 Streptavidin Dynabeads (Life Technologies) were washedwere added to 3 ml of diluted chromatin, which was proteasemixed inhibitor,by end-to-end and 0.1rotation U/ NaCl, 1% SDS, 50 mM Tris, pH 7.0, 1 mM EDTA, 15% formamide, 1 mM DTT, Chromatin was diluted in two times volume with hybridization buffer (750 mM mostchromatin had solubilized and DNA was in the size range of 100–500 bp. U/ tor,0.1 and EDTA,DTT,SDS;andmM freshly1%with1 added complete protease inhibi mM 10 Tris,7.0, mM pH (50 buffer lysisnuclear in lysed further Nucleiwere suspensions Cell ice.were homogenizedthen at and2,500pelleted DTT, complete protease inhibitor, and 0.1 U/ pH 7.0, 10 mM KOAc, and 15 mM MgOAc, with freshly added 1%Tris, M (0.1 swellingbufferNP-40,resuspended inand werepelleted cells The min. 5 1 mM for glycine M 0.125 with quenched then was cross-linking and temperature, cross-linked with 1% glutaraldehyde or formaldehyde in PBS for 10 min at modifications someroom with described, previously RNA. of pulldown Biotin-oligo 50. 49. 48. 47. 46. F ends, with error bars showing s.e.m., if applicable. After analysis of variance by independentexperiments or the actual number of cells stated in the figure leg Statistical analysis. Ct value >35; Ct values for all inputs were 21–23. tions. In real-time PCR data shown in the figures, ND (not detected) refers to a PCR cycle numbers were set between 20 and 25 to avoid saturation of PCR reac to standard procedures. For semiquantitative PCR and semiquantitative(Invitrogen) RT-PCR, on a PikoReal Real-Time PCR System (Thermo Scientific)PCR (qPCR) was performedaccording with Platinum SYBR Green qPCR Supermix UDG thesupplied protocol, with random hexamer primers or oligo dT. Quantitative TranscriptionRNAReverseGoScript the with System (Promega) according to for quantitative reverse-transcription PCR (qRT-PCR) was synthesized(Invitrogen) accordingfrom to totalthe manufacturer’s procedures. Complementary DNA reactions. PCR test, the statistical significance and

Chu, C., Qu, K., Zhong, F.L., Artandi, S.E. & Chang, H.Y. Genomic maps of long of maps Genomic H.Y. Chang, & S.E. F.L.,Artandi, Zhong, K., Qu, C., Chu, Listerman, I., Sapra, A.K. & Neugebauer, K.M. Cotranscriptional coupling of splicing S. Guang, Kent, W.J. Y. Luo, ocdn RA cuac rva picpe o RAcrmtn interactions. RNA-chromatin of principles reveal Cell Mol. occupancy RNA noncoding cells. mammalian in splicing Biol. Mol. Struct. RNA Nat. messenger precursor and recruitment factor transcription. of phase elongation (2002). (2010). cells. stem/progenitor neural adult of differentiation t al. et

44 et al. t al. et µ , 667–678 (2011). 667–678 , l RNase inhibitor) on ice for 10 min and were sonicated until sonicatedwere and min 10 for ice inhibitor)onRNase l oa RA a etatd rm el wt TIo reagent TRIzol with cells from extracted was RNA Total rgl X etl eadto poen euae poieain and proliferation regulates protein retardation mental X Fragile The human genome browser at UCSC. ml rgltr RA ihbt N plmrs I drn the during II polymerase RNA inhibit RNAs regulatory Small The values reported in the graphs represent averages of three nature structural & molecular biology molecular & structural nature 13 , 815–822 (2006). 815–822 , µ l RNase inhibitor). Biotin-DNA oligos (100 pmol) itnoio ulon a crid u as out carried was pulldown Biotin-oligo Nature P value were evaluated by Student’s

465 µ , 1097–1101 (2010). 1097–1101 , l RNase inhibitor) for 10 min on 5 0 . Briefly, log-phase cells were cells Briefly,log-phase . Genome Res. LS Genet. PLoS µ l washed/blockedl

12 6 µ g e1000898 , l yeast total , 996–1006 for 5 min.for 5 t test. - - -