SUPPLEMENTARY INFORMATION

Transcription

1 SUPPLEMENTARY INFORMATION doi:.8/nture99 Supplementry Tle : Primers nd proes. Sequences re written in 5 - direction. Vector construction cirs-7 forwrd cirs-7 reverse cirs-7ir forwrd cirs-7ir reverse cirs-7fs forwrd cirs-7fs reverse Sry forwrd Sry reverse mir-7- forwrd mir-7- reverse mir-8 forwrd mir-8 reverse mir-7-psicheck forwrd mir-7-psicheck reverse cirs-7-psicheck forwrd cirs-7-psicheck reverse Sry-psiCheck forwrd Sry-psiCheck reverse EGFR-psiCheck forwrd EGFR-psiCheck reverse GACCCAAGCTTCTGTAAGAGTAGTCTCATGATGT TTCAGGCGGCCGCCAAACTGCAGTACTGTTGGTTCAT TAGAACTCGAGCTGTAAGAGTAGTCTCATGATGT TAGAACTCGAGTGGTTACTCTGGACATTGAT TTCAGAAGCTTAGGGTTTCCGATGGCACCT TAGAACTCGAGCTGGATATTGCAGACACTGG TTCAGGGATCCTGGACTAGGGAGGTCCTGAA TAGAAGCGGCCGCAACCCAAAAGGAAGGAGCAG ATAGAAGCGGCCGCGCCATGGTGTCTCAACCTTT ATAGAAGTCGACGCAATGTTAAGTGTCAAGAAAAATG ATAGAAGCGGCCGCGTCATCCTGTCTCCCCTCCT TAGAAGTCGACGGTTTCCTCACAGGCAGGT TAGAACTCGAGACAACAAAATCACTAGTCTTCCATCTAGAATGAC GTCATTCTAGATGGAAGACTAGTGATTTTGTTGTCTCGAGTTCTA TTCAGCTCGAGGGTTTCCGATGGCACCTG TTCAGGCGGCCGCCTGGATATTGCAGACACTG TAGAACTCGAGCATTTCAGATCTTGATTTTTAGTGT GTCATGCTAGCCAGTGATGTCAGCTGTTAGTAAGT AATTGCGGCCGCCACGGAGGATAGTATGAGC AATTTCTAGATTCATTGAGACAAAAATCAAATAC qpcr / RT-PCR cirs-7 forwrd cirs-7 reverse cirs-7 (2) forwrd GAPDH forwrd GAPDH reverse Sry forwrd Sry reverse SNCA forwrd SNCA reverse EGFR forwrd EGFR reverse IRS2 forwrd IRS2 reverse Oligo-dTN RACE reverse Proes mir-7 proe mir-769 proe mir-8 proe mir-5 proe cirs-7 proe Sry proe 8S proe cirs-7 (hs) FISH proe (mino-llyl-t underlined) cirs-7 (mmu) FISH proe (mino-llyl-t underlined) cirs-7 (mmu) AP-proe mir-7 FISH/AP-proe (LNA underlined) mir-449 FISH Proe (LNA underlined) Gpdh AP-proe sirna / mirna mir-8 iotin guide mir-8 pssenger mir-7 iotin guide mir-7 pssenger ACGTCTCCAGTGTGCTGA CTTGACACAGGTGCCATC ATAGCTTCCGAAAAATCCAG GTCAGCCGCATCTTCTTTTG GCGCCCAATACGACCAAATC AGTTCCACGACCAGCAGCT CAGCTGCTTGCTGATCTCTGTA CTGCTGCTGAGAAAACCA CCTTGGTTTTGGAGCCTA CGAGGGCAAATACAGCTT GCCGTCTTCCTCCATCT TCTTGTCCCACCACTTGA CAGTGCTGAGCGTCTTCT GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN GCGAGCACAGAATTAATACGACT ACAACAAAATCACTAGTCTTCCA AGCTCAGAACCCAGAGGTCTCA CGGCCTGATTCACAACACCAGCT TGTAAACCATGATGTGCTGCTA TTGGAAGACTTGAAGTCGCTGGAAGACCCGGAGTTGTTGGAAGACCTTGACACAGGTGCC GGCTGCCAATAAAAGCTTTGCTGGTTTTTGGAGTACAGGTGTGCAGCTCTACTCCAGTCT TTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATGGATCCTCGTTAAAGGATTTAAAGTGGACTCATTCCAATTACAG TACATGGATTTGTTGGAAGACATGGATTTTCTGGAAGACATGGATTTTCT TTCTGGGAAGACTTGGATTTTCGGGAAGACTTGGATTTTCGGGAAGACTT TGGGAAGACTCGGATTTCTGGGAAGACT ACAACAAAATCACTAGTCTTCCA ACCAGCTAACAATACACTGCCA CCTGCTTCACCACCTTCTTGATGTCA AGCTGGTGTTGTGAATCAGGCCG[Btn] GCCTGATTCACAACACCAGCGCC TGGAAGACTAGTGATTTTGTTGT[Btn] CAACAAAATCACTAGTCTACCTAA

2 RESEARCH SUPPLEMENTARY INFORMATION 8mer: GUCUUCCA CAGAAGGU 5 7mer m8: GUCUUCC CAGAAGGU 5 7mer A: UCUUCCA AGAAGGU 5 mir 67: CUUCCAGCAUCUCCAGGGCUUCC : : GAGGUCGGGGAGGUCCCGAAGGA 5 d dg / Trget length 77 sites; E=.6e 299 c mir 67 mir 67 Supplementry Figure : Evolutionry conservtion of cirs-7., Orgniztion of cirs-7 locus in selected mmmlin species. mir-7 nd mir-67 trget sites re denoted ccording to legend shown to the right. mir-67 trget site is defined s the 22mer, TTCCAGCATCTCCAGGGCTTCC, llowing mismtch. The oundries of cirs-7 (lck horizontl r) is sed on the position of conserved splice donor (TCCAGGTATT) nd splice cceptor (TCCAAT[AG]TC[CT]AGGG, optionl nucleotides re shown in rckets) sites. The crinkled lines represent gps in the genome ssemly., Welogo of the MEME computed highest rnking 6-7mer occurring etween 2 nd times within humn cirs-7. The motif is depicted with se piring potentil to mir-7. Not ll sites predicted y MEME hve perfect seed sequences, explining the discrepncy etween the 77 sites predicted y MEME nd the 7 seed sites depicted in () c, Welogo of the identified mir-67 trget sites from ll plcentl mmmls investigted, s represented y non-filled oxes in () (except elephnt, where no mir-67 sites ws found) depicted with se piring potentil to mir-67. d, Boxplot showing thermodynmic predictions using MultiRNAFold (ver. 2 ) of 7 humn mir-7 trget sites (7mers) upon se piring with mir-7 nd the mir-67 trget site (22mer) upon se piring with mir-67 normlised to trget site length. 2

3 SUPPLEMENTARY INFORMATION RESEARCH nt HITS CLIP Conservtion mirna trget sites SA SD Non liner splice junction Non liner splice junction GGTGTCTGCCGTATCCAGGGTTTCCAGTGGTGCCAGTACCAAGGTCTT...CAGGGTTTCCAGTGGTGCCAGTACCAAGGTCT. 4...CAGGGTTTCCAGTGGTGCCAGTACCAAG......CAGGGTTTTCAGTGGTGCCAGTACCAAGGTCT....TAGGGTTTCCAGTGGTGCCAGTACCAAGGTCT. *...GGGTTTCCAGTGGTGCCAGTACCAAGGTC.. #...TATCCAGGGTTTCCAGTGGTGCCAGTACCAAGGTCT. TTCAGGTTTTCTGGTGTCTGCCGTATCCAGGGTTTCCAGTGGTGCCAGTACC...TTTCTGGTGTCTGCCGTATCCAGGGTTTCCAGTG TTTTCTGGTGTCTGCCGTATCCAGGGTTTCCAGTG TTTTCTGGTGTCTGCCGTATCCAGGGTTTCCA.....CAGGTTTTCTGGTGTCTGCCGTATCCAGGGTTTCCA......TTTCTGGTGTCTGCCGTATCCAGGGCTTCCAGTG......TTTCTGGTGTCTGCCGTATCCAGGGTTTCCAGTC......TTTCTGGTGTCTGCCGTATCCAGGGTTTCCAGTGGA......TTTCTGGTGTCTGCCGTATCCAGGGTTTCCAGTGTT... Supplementry Figure 2: HITS-CLIP reds derived from non-liner RNA., Schemtic representtion of the genomic locus encoding the murine cirs-7 with predicted mir- 7 nd mir-67 trget sites (s in Fig. ) nd mmmlin conservtion scores. Distriution of Ago2 HITS-CLIP reds from mouse rin is shown ove., All reds from Ago2 HITS-CLIP were trimmed to 5 nt from the 5 - or -end, generting set of 5-mers reds reflecting the end or 5 end of ech red, respectively. Susequently, the ends were mpped towrds the 5 end of murine cirs-7 (lck sequence), wheres the 5 ends of ll reds (5mers) were mpped towrds the end of murine cirs-7 (grey sequence). For ech perfectly mpped 5-mer (underlined), the untrimmed red ws recovered nd ligned to non-liner spliced cirs-7 sequence (lck-grey chimeric sequence). Red count is depicted to the right of ech sequence. (*) denotes sequence not in support of non-liner splicing of cirs-7. (#) denotes miguous sequence.

4 RESEARCH SUPPLEMENTARY INFORMATION. TAPEXO RNAseR TAPEXO RNAseR TAPEXO RNAseR.... / GAPDH EV fs ir k 2 4 c EV fs TAP EXO: RNAse R: GAPDH 8S EV CiRS 7 fs CiRS 7 Supplementry Figure : Circulriztion of cirs-7., RT-PCR on RNA from HEK29 cells trnsfected with pcdna (EV), pcdna-cirs-7-fs, pcdna-cirs-7-ir, nd pcdna- cirs-7, s denoted ove, using outwrds fcing primers (cirs-7 (2) forwrd / cirs-7 reverse) s depicted. The upper nd lower nd corresponds to inclusion or exclusion of previously identified intron 4 s indicted to the right., c, Northern lot () or qrt-pcr (c) on RNA from HEK29 cells trnsfected with empty vector (pcdna; EV), pcdna- cirs-7-fs, nd pcdna-cirs-7. Prior to nlysis, the RNA ws either digested with Tocco Acid Pyrophosphte tht removes the 5 cp followed y Termintor 5 Exonuclese tretment (TAP-EXO), or with RNse R, s indicted. Error rs represent s.d. (n=4). 4

5 SUPPLEMENTARY INFORMATION RESEARCH EV fs ir k RT: * AAAAAAAAA AAAAAAAAA *.6.4 fs Reltive RNA levels.2 69 Supplementry Figure 4: Liner version of cirs-7 is sensitive towrds mir-7., RT-PCR on RNA from HEK29 cells trnsfected with pcdna (EV), pcdna-cirs-7-fs, pcdna- cirs-7-ir, nd pcdna-cirs-7 using the RACE pproch. The upper nd lower nd corresponds to inclusion or exclusion of previously identified intron 4 s indicted to the right., Quntifiction of cirs-7 levels derived from trnsfections with pcdna-cirs-7-fs nd pcdna-cirs-7 long with plsmids expressing either mir-7 (pjebb-7) or mir-769 (pjebb-769) sed on northern lot signl intensities. Error rs represent s.e.m (n=). *, p<

6 RESEARCH SUPPLEMENTARY INFORMATION trsa ppp ppp Pellet INPUT trsa trsa kd α AGO2 α HuR Pellet / INPUT INPUT trsa trsa+ Pellet c Pellet INPUT trsa trsa+ trsatrsa Supplementry Figure 5: Streptvidin-ptmer cpture of AGO2 nd mir-7.,, T7-trnscripts covering the entire cirs-7 sequence with or without streptvidin ptmer (trsa) sequence in the 5 end (s schemticlly depicted ove), were incuted with cell lyste pre-trnsfected with either mir-7 (pjebb-7) or mir-769 (pjebb-769) expression vectors. After streptvidin cpture, the input nd ound frction were nlysed y western lot using nti-ago2 ntiody (α-ago2) or nti-hur (control; α -HuR) () or y mirna tqmn qpcr detecting mir-7 (drk rs) or mir-769 s control (light grey rs) normlised to RNU48 nd INPUT (). Error rs represent s.d. (n=). c, Northern lot of T7-trnscried cirs-7 with (trsa+) or without trsa sequence (trsa-) from prior (INPUT; T7 trnscripts lone) nd fter cell lyste incution nd streptvidin cpture (Pellet). 6

7 SUPPLEMENTARY INFORMATION RESEARCH merged c merged + DCPA merged + Supplementry Figure 6: Intrcellulr locliztion of cirs-7 nd mir-7.,, RNA-FISH nd IF-FISH on HeL cells trnsfected with constructs s indicted (s in Fig 2c-d). c, RNA-FISH on primry cells extrcted from mouse rin using Cy-lelled cirs-7 proe (left pnel), Cy5-lelled murine mir-7 proe (middle pnel) nd merged (right pnel). Scle rs, 5 µm. 7

8 8 W W W. N A T U R E. C O M / N A T U R E VI V IV % co expression e c +/+ *** /+ CG R CG L CG 2R CG 2L CG R CG L Men ± SD / Co expression +/+ % ± 2 merged No co expression /+ % ± Supplementry Figure 7: Su-rin expression of cirs-7 nd mir-7 in mouse., Mouse rins sections hyridized with lkline phosphtse-coupled proes specific for GAPDH mrna (top pnel) or cirs-7 (ottom pnel). HP, hippocmpus; NCx, neocortex; Th, thlmus., cirs-7 is expressed in the neuronl cell ody (rrowheds) nd dendrites (rrows), here shown for lyer IV-VI of somtosensory cortex in 5μm thick section. c, Doule fluorescence in situ hyridiztion showing co-expression etween cirs-7 (left pnel) nd mir-7 (middle pnel), merged imge (right pnel) visulized y confocl microscopy. d, In situ hyridiztion for cirs-7 (left pnel) nd mir-7 (right pnel) performed on djcent, 5 μm thick sections, which llows identifiction of co-expressing neurons (rrows), here shown for the dentte hilus (h). For presenttion one of the digitlized imges ws lterlly reversed. e, Quntittive dt on % co-expression etween cirs-7 nd mir-7 in cingulte gyrus (CG) lyer IV-V. Tle shows dt otined from right (R) nd left (L) CG. Dt re presented s men ± SD. ***, p <.. Br: mm (), μm (), μm (c) nd 5 μm (d). d Gpdh Supplementry Figure 7 RESEARCH SUPPLEMENTARY INFORMATION

9 SUPPLEMENTARY INFORMATION RESEARCH EGFR UTR RL RL.5 *** *** RL / FL RL / FL EV EV N.C. 8:4:2 4:4:2 2:4:2 :4:2 ::psicheck rtios Supplementry Figure 8: Luciferse reporter ssy., The UTR of mir-7 trget gene, EGFR, ws inserted into psicheck vector (schemtic representtion of trget site depicted ove). HEK29 cells in 2-well pltes were co-trnsfected with.5 µg empty vector (EV) or pcdna-cirs-7 together with nm mir-7 mimic sirna or nm N.C. (negtive control sirna) nd. µg psicheck reporter plsmid contining the entire EGFR UTR. Forty eight hours fter trnsfection, reltive luminescence ws mesured nd plotted., Vectors expressing cirs-7 (pcdna-cirs-7), mir-7 (pjebb-7) nd psicheck-cirs-7 were trnsfected in vrious plsmid weight rtios s indicted. Forty eight hours fter trnsfection, reltive luminescence ws mesured nd plotted. Error rs represent s.d. (n=). ***, p<.. 9

10 RESEARCH SUPPLEMENTARY INFORMATION HeL EV HeL N.C. mir 67 N.C. mir 67.E+.E+ *.E.E 2.E.E 4 8S / GAPDH.E HeL EV HeL HEK29 Brin.E 6 c d 2.E 2.E 2.E 2 5.E EV 8.E 6.E 4.E / GAPDH 2.E N.C. mir 67 / GAPDH.E+.E (nm) (nm) Supplementry Figure 9: Stle expression of cirs-7 in HeL cells., qrt-pcr on RNA purified from HeL-EV, HeL-ciRS-7, HEK29 cells, nd totl rin quntifying cirs-7 levels reltive to GAPDH mrna., Northern lot using μg RNA from HeL-EV (with empty vector expression cssette) or Hel-ciRS-7 (with cirs-7 expression cssette). Cells were trnsfected with nm non-relted control (N.C.), mir-7 or mir-67 mimic sirnas s indicted. The lot ws proed with cirs-7 nd 8S (loding control). The sterix (*) denotes high-moleculr, non-circulr RNA species produced from the cirs-7 construct. c, d, RT-qPCR quntifiction of cirs-7 in HeL-ciRS-7 (light grey line) or HeL-EV cells (drk grey line) trnsfected with grdient concentrtion of mir-7 (c) or in HeL-ciRS-7 cells trnsfected with mir-67 mimic sirna (light grey line) or N.C. (negtive control; drk grey line) sirna 24 hrs prior to trnsfections with mir-7 grdient (d). Error rs represent s.d. (n=4).

11 SUPPLEMENTARY INFORMATION RESEARCH Sry c SA SD EV Sry SA SD * EV Sry +RT RT +RT RT Sry 8S 5p Supplementry Figure : Sponging of mir-8 y Sry circrna expression., Schemtic representtion of Sry expression vector (pcdna-sry) in which the tet-responsive CMV promoter from the pcdna5/frt/to vector hs een inserted in n inverted orienttion downstrem the Sry locus., Northern lot using μg RNA from HEK29 cells trnsfected with either empty vector (EV, pcdna) or the Sry expression construct (pcdna-sry). Blot ws proed with Sry nd 8S (upper nd lower pnel, respectively). Position of the circulr Sry RNA is indicted. The sterix (*) denotes high-moleculr, non-circulr RNA species produced from the Sry construct. c, RT-PCR on RNA from HEK29 cells trnsfected with either empty vector (EV, pcdna) or Sry expression vector (pcdna-sry) using primers depicted with rrows ove. RT rections were performed with (+RT) or without reverse trnscriptse (-RT) s control.

12 RESEARCH SUPPLEMENTARY INFORMATION References. Biley, T. L. & Elkn, C. Fitting mixture model y expecttion mximiztion to discover motifs in iopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2, 28-6 (994). 2. Andronescu, M., Zhng, Z. C. & Condon, A. Secondry structure prediction of intercting RNA molecules. J. Mol. Biol. 45, 987- (25).. Chi, S. W., Zng, J. B., Mele, A. & Drnell, R. B. Argonute HITS-CLIP decodes microrna-mrna interction mps. Nture 46, (29). 4. Hnsen, T. B. et l. mirna-dependent gene silencing involving Ago2-medited clevge of circulr ntisense e RNA. EMBO J., (2). 2