Roquin binds inducible costimulator mrna and effectors of mrna decay to induce micrornaindependent post-transcriptional repression

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1 ins inuile ostimultor mrna n effetors of mrna ey to inue mirornainepenent post-trnsriptionl repression Elke Glsmher 1, Ki P Hoefig 1,3, Kthrin U Vogel 1,3, Niol Rth 1, Lirui Du 1, Christine Wolf 1, Eliseth Kremmer 1, Xiozhong Wng 2 & Vigo Heissmeyer 1 Nture Ameri, In. All rights reserve. The moleulr mehnism y whih roquin ontrols the expression of inuile ostimultor () to prevent utoimmunity remins unsolve. Here we show tht in helper T ells, roquin lolize to proessing (P) oies n ownregulte expression. The repression ws epenent on the RNA helise, n roquin interte with n the enhner of epping E4, whih t together in mrna epping. Sequenes in roquin tht onfer P-oy loliztion were essentil for roquin-meite repression. However, this proess i not require mirornas or the RNA-inue silening omplex (RISC). Inste, roquin oun mrna iretly, showing n intrinsi preferene for previously unreognize sequene in the 3 untrnslte region (3 UTR). Our results support moel in whih roquin ontrols expression through ining to the 3 UTR of mrna n y interting with proteins tht onfer post-trnsriptionl repression. is CCCH-type zin finger protein tht estilizes the mrna of the inuile ostimultor () in proess tht requires the 3 untrnslte region (3 UTR) of mrna 1. Loss of this post-trnsriptionl regultion uses utoimmunity in R3h1 sn/sn mie, whih re homozygous for point muttion in R3h1, the gene tht enoes roquin 1,2. How mrna is reognize y roquin protein n how this reognition inues mrna ey is unknown t present. Post-trnsriptionl regultion of gene expression ontrols key eisions of innte n ptive immune responses 35. Pthwys involve in trnsltionl repression n mrna ey use mny ifferent moleulr mehnisms ut uniformly epen on trns-ting ftors tht in to is-regultory elements in trget mrnas. One lss of trnsting ftors re mirornas (mirnas) tht, when loe onto the RNA-inue silening omplex (RISC), n se pir with prtilly omplementry sequenes in the 3 UTR of trget mrnas 5. RNA-ining proteins represent nother lss of trns-ting ftors. Cis elements for these ftors hve een esrie in single-strne RNA; however, they re not well efine n the reognition is thought to epen on shpe s well s sequene 3,6. Trns-ting RNA-ining proteins n mirnas n trget the sme mrna. The ifferent trns-ting ftors n oopertively repress the trget 7 or the RNA-ining protein n lok mirna-epenent repression 8. In ition, trns-ting protein ftors t together with mirnas n entrl omponents of the RISC, the rgonute (Ago) proteins, in n opposing progrm n ugment trnsltion 9. Whether the vrious pthwys of post-trnsriptionl regultion t together in generl or work in prllel n onverge uner speifi ellulr onitions nees further investigtion n requires geneti elinetion of the moleules involve. A 3 UTR region of mrna hs een esrie s eing importnt for roquin-meite repression of 1. On the sis of the investigtion of puttive mir-1-ining site in this region, funtionl epenene of roquin on mirna hs een suggeste 1. However, it remins unler whether the mirna itself or roquinmirna or roquinmirna-inue silening omplex (mirisc) omplex is the trns-ting ftor tht enles reognition of the mrna n repression of ellulr. expression is sustntilly inue on the surfe of T ells fter reognition of ntigen. Its trnsription is upregulte erly fter triggering of the T ell ntigen reeptor n in response to tivtion of the trnsription ftor NFAT. However, the lrgest mounts of protein pper with temporl ely, whih emonstrtes pronoune post-trnsriptionl regultion. The inue expression regultes ytokine proution in T ells, whih llows them to provie B ells help for the proution of high-ffinity ntioies in the germinl enter retion R3h1 sn/sn mie tht express mutnt roquin proteins show spontneous germinl enter formtion n evelop utoimmune phenotypes similr to systemi lupus erythemtosus in humn ptients 2. Consistent with their errnt high expression in T ells, these mie hve more folliulr helper T ells 1,2,16. However, severl utoimmunity-ssoite phenotypes ispper in R3h1 sn/sn mie 1 Helmholtz Zentrum Münhen, Germn Reserh Center for Environmentl Helth, Institute of Moleulr Immunology, Munih, Germny. 2 Deprtment of Biohemistry, Moleulr Biology n Cell Biology, Northwestern University, Evnston, Illinois, USA. 3 These uthors ontriute eqully to this work. Corresponene shoul e resse to V.H. (vigo.heissmeyer@helmholtz-muenhen.e). Reeive 16 April; epte 14 June; pulishe online 18 July ; oi:.38/ni.192 nture immunology VOLUME 11 NUMBER 8 AUGUST 725

2 Nture Ameri, In. All rights reserve. Antiroquin Antituulin GFP GFP hi Tuulin CD4 2 1 GFP sh-c4 Figure 1 expression is ple uner the ontrol of roquin in ifferentite helper T ells. () Immunolot nlysis of roquin n tuulin (loing ontrol) in CD4 T ells from Tg(DO11.)- Tg(CARΔ-1) mie infete ex vivo with enovirl vetors enoing ontrol shrna () or shrna speifi for R3h1 () n stimulte with plte-oun nti-cd3 n nti-cd28 uner T H onitions efore eing sorte for GFP expression y flow ytometry. () Expression of n CD4 in ells trnsue with vetor enoing ontrol shrna or shrna speifi for R3h1 () or heterozygous for Ios efiieny 1, whih inites tht is the ritil trget in roquin-meite prevention of lupus-like utoimmunity. The R3h1 sn/sn muttion is positione in the ROQ omin of roquin n reners it less effetive in ontrolling expression 1. The integrity of the ROQ omin is therefore essentil in the prevention of utoimmunity; however, the moleulr funtion of this omin remins unler. Here we show tht roquin is the trnsting ftor tht trgets mrna for post-trnsriptionl repression. oun RNA through its ROQ omin n CCCH-type zin finger n reognize region in the 3 UTR of. lso interte with proessing (P)-oy ftors, whih re involve in mrna ey, n ws le to repress even in the omplete sene of ellulr mirnas or RISC formtion. RESULTS expression is ontrolle y roquin To investigte the ontriution of roquin to regultion in ifferentite helper T ells, we estlishe n enovirl knokown pproh. We use primry CD4 T ells from Tg(DO11.)- Tg(CARΔ-1) mie to introue enovirl vetors 17. T ells from these mie re permissive for enovirl infetion through trnsgeni expression of signling-intive version of the humn oxskie enovirus reeptor. We infete CD4 T ells efore tivting them with ntioy to CD3 (nti-cd3) n nti-cd28 in ulture onitions tht skew helper T ell ifferentition (Fig. 1). By using enovirl vetors oexpressing green fluoresent protein (GFP) with short hirpin RNA (shrna) speifi for R3h1 or the ontrol gene C4, we were le to ownregulte the expression of roquin protein (Fig. 1) n R3h1 mrna (Fig. 1,g). However, we oserve ownregultion of roquin protein only in ells with the highest expression of GFP (25%), whih orrelte with the oserve lower effiieny of RNA-meite interferene in primry CD4 T ells 18. Knokown of roquin resulte in more protein n Ios mrna in T helper mrna (%) mrna etetion e f g (MFI 2 ) Ios R3h2 R3h1 Cells (%) Ios mrna (reltive) T H R3h1 mrna (reltive) type (T H ), T H 1 n T H 2 ells (Fig. 1f). The effet ws speifi, s it i not ffet the expression of CD4 protein (Fig. 1) or R3h2 mrna (Fig. 1), the prlog of R3h1 (ref. 19). In ition, expression ws not inue y expression of n empty shrna vetor or y knokown of CD4 (Fig. 1). Quntifition of the surfe expression of protein (Fig. 1,e) n Ios mrna (Fig. 1f) uring the tivtion n ifferentition of CD4 T ells uner T H, T H 1 n T H 2 onitions emonstrte tht protein n Ios mrna inrese from T H ells to T H 1 ells to T H 2 ells (Fig. 1f), s reporte efore. Regrless of the ifferentition onitions, there ws signifint inrese in surfe expression in ells trnsue with shrna speifi for R3h1 (Fig. 1,e). We oserve similr inrese in Ios mrna in the ulk popultion ontining 729% GFP ells (Fig. 1f). The knokown iminishe R3h1 mrna to 4675% of enogenous mounts in the ulk popultion (Fig. 1g). These finings show tht the expression of is ple uner the post-trnsriptionl ontrol of roquin in helper T ells. interts with the P-oy pthwy We use eletion mutgenesis to ientify regions in roquin ritil for the repression esrie ove. We nlyze the mutnts in mouse emryoni firolsts (MEFs) first trnsue with retrovirus expressing full-length humn mrna n susequently superinfete with retrovirus ontining n internl riosoml entry site oexpressing the mrker n wil-type or mutnt versions of roquin. Truntion of the roxyl terminus in mutnt roquin(159), whih onsists of mino is 159 n lks the proline-rih n the oile-oil regions, intivte roquin-meite repression of (Fig. 2). In ontrst, roquin(1951), mutnt lking only the roxy-terminl oile-oil omin, ws slightly less tive thn wil-type roquin (Fig. 2). The intive roquin(159) mutnt fuse to GFP showe errnt iffuse ytoplsmi loliztion fter trnsfetion into HEK293 humn T H T H 1 T H 2 T H T H 1 T H 2 T H T H 1 T H 2 C4 (sh-c4) n stimulte uner T H 2 onitions. () RT-PCR nlysis of the expression of Ios, R3h2 n R3h1 mrna in the frtion of ells with the highest expression of GFP (%), sorte from ells trnsue with R3h1-speifi shrna n stimulte uner T H 1 onitions, presente reltive to the expression in ells trnsue with ontrol shrna, set s %. P =.2 n P =.1 (one-wy nlysis of vrine (ANOVA)). (,e) expression in gte GFP ells infete with enovirl vetor enoing ontrol or R3h1-speifi shrna n stimulte with nti-cd3 n nti-cd28 uner T H, T H 1 or T H 2 onitions. MFI, men fluoresene intensity. P =.35, T H ; P =.23, T H 1; P =.29, T H 2 (one-wy ANOVA). (f,g) RT-PCR of Ios mrna (f) or R3h1 mrna (g) expression in ulk popultions of ells trnsue with ontrol or R3h1-speifi shrna, ontining 729% GFP ells fter stimultion with nti-cd3-cd28 uner T H, T H 1 or T H 2 onitions, presente reltive to expression in ells trnsue with in T H onitions (f) or with in T H 1 onitions (g), set s 1.. Dt re from one experiment representtive of three (,f,g; error rs (,f,g), normlize errors fter effiieny orretion se on reltive stnr urves) or re from three inepenent experiments (e; verge n s..). T H 1 T H VOLUME 11 NUMBER 8 AUGUST nture immunology

3 Nture Ameri, In. All rights reserve. Figure 2 Croxy-terminl sequenes in roquin re require for repression n roquin loliztion. () Domin orgniztion of roquin, inluing the RING finger, ROQ omin, zin finger, region rih in proline, n the oile-oil omin. Numers ove inite mino i positions use s mino- or roxyterminl ens of eletion mutnts. () Expression of n roquin (ssesse s ) in MEFs trnsue with retrovirus enoing full-length humn mrna n superinfete with retrovirus ontining n internl riosoml entry site n (IRES) lone or with wil-type roquin (roquin(wt)) or mutnt roquin (roquin(1951)) or roquin(159)). Dt re representtive of three experiments. () expression in the ells in, presente reltive to its expression in ontrol ells (empty vetor), set s %. P =.27 n P =.8 (one-wy ANOVA). Dt re from three inepenent experiments (verge n s..). () Confol mirosopy of the ololiztion of (lue) n FMRP (re) with GFP-tgge wil-type roquin (GFP-roquin; green) in primry CD4 T ells with (Arsenite) n without (No stress) rsenite tretment fter 48 h of stimultion with plte-oun nti-cd3 n nti-cd28. White rrowhes inite -, GFP- n FMRP-lele foi. Zoom (fr right), enlrgement of imges t left. Originl mgnifition, 232 (min imges) or 262 (fr right). Dt re representtive of two experiments. emryoni kiney ells (Supplementry Fig. 1), in ontrst to the loliztion of fusion proteins of GFP n roquin(1951) or wiltype roquin, whih lolize to ytoplsmi foi n showe little iffuse ytoplsmi stining (Supplementry Fig. 1,). We ssesse roquin loliztion in primry CD4 T ells trnsue with enoviruses expressing GFPwil-type roquin (Fig. 2 n Supplementry Fig. 1) n HEK293 ells trnsfete with GFPwil-type roquin (Supplementry Fig. 1) y ostining with ntioies tht roustly ientify P oies or stress grnules. Mny of the roquin-enrihe foi in HEK293 or CD4 T ells showe full ololiztion with the RNA helise n we therefore ientifie them s P oies. In ontrst, FMRP, mrker protein of stress grnules 21,22, showe iffuse stining in oth ell types in the presene or sene of etopi roquin expression (Fig. 2 n Supplementry Fig. 1). Consistent with tht, stining with n ntioy to the stress grnule mrker G3BP1 onfirme the sene of stress grnules in CD4 T ells fter expression of roquin (Supplementry Fig. 1). Quntifition of loliztion of GFP-roquin fusion proteins in CD4 T ells showe tht in 62% of the ells, roquin ws ssoite with P oies, wheres it lolize to the stress grnules in only 1.7% of the ells. There ws no effet of roquin expression on the perentge of ells with P oies (68% of ll ells) or stress grnules (18% of ll ells) or on the numer of P oies or stress grnules per ell (t not shown). Sujeting CD4 T ells to rsenite-inue stress (Fig. 2) inue the formtion of stress grnules in 85% of ells, n 89% of ells expressing GFP-roquin showe n ssoition of roquin with stress grnules. remine ssoite with P oies in only 5.5% of these ells. Experimentl inution of stress hnge neither the perentge of ells positive for P oies nor the numer of P oies per ell (on verge, 2) or stress grnules per ell (on verge, 2.75; t not shown). Similrly, in HEK293 ells, the ololiztion of roquin n ws nerly ompletely rogte in ells trete with rsenite to 2 1 No stress Arsenite ,13 IRES- RING ROQ Zin Proline-rih Coile-oil (WT) IRES (1951) IRES (159) IRES inue oxitive stress (Supplementry Fig. 1). During ggregtion of the messenger rionuleoprotein omplexes tht form P oies, sequenes rih in glutmine n sprgine meite protein-protein intertions The roxy-terminl region of roquin showe greter frequeny of glutmine n sprgine resiues in sequenes jent to the proline-rih region 25. Artifiilly efine frmes of mino is reh mximum ontent of 17 glutmine n sprgine resiues in mouse roquin, with 7.68 s the preite verge 25. We therefore investigte loliztion n funtion of the mutnt roquin(1749), whih ontins the proline-rih region ut lks the sequenes enrihe in glutmine n sprgine resiues. This mutnt h impire foi formtion in the sene of stress (Fig. 3) ut ws still le to trnslote into stress grnules fter rsenite tretment (Fig. 3). Overexpression of this mutnt interfere with P-oy formtion, s juge y the iffuse ytoplsmi loliztion of (Fig. 3). However, fter the inution of stress, the -lele foi quikly reppere (Fig. 3). In funtionl ssys, roquin(1749) ws onsierly impire in its ility to ownregulte fter sequentil infetion of the mouse firolst ell line NIH3T3 (Fig. 3,). These finings positively orrelte roquin funtion with its loliztion to P oies ut not with its loliztion to stress grnules. To investigte the possiility tht stress grnules re ispensle, we teste roquin funtion in the sene of T ell intrellulr ntigen 1 (TIA-1; Supplementry Fig. 2). TIA-1-efiient MEFs hve impire ssemly of stress grnules, s etermine y stining for the stress grnule mrkers TIAR, eif3 n G3BP 26. In TIA-1-efiient n ontrol MEFs (Fig. 3) sequentilly trnsue with retrovirl n roquin onstruts, there ws lmost no effet of Ti1 eletion on the ility of roquin to repress (Fig. 3,e). The t suggest tht t lest in the sene of ell stress, roquin-meite repression of oes not epen on TIA-1 funtion n inste requires physil or funtionl intertions with P-oy omponents. GFP-roquin FMRP Overly expression (%) Control (WT) (1951) (159) GFProquin Zoom GFProquin FMRP Overly FMRP Overly nture immunology VOLUME 11 NUMBER 8 AUGUST 727

4 Nture Ameri, In. All rights reserve. No stress Arsenite GFProquin(1749) FMRP Overly Zoom Figure 3 -meite repression of orreltes with P-oy loliztion inepenently of TIA-1. () Confol mirosopy of the ololiztion of (lue) n FMRP (re) with GFP-tgge mutnt roquin(1749) (green) in HEK293 ells with n without rsenite tretment. White rrowhes inite -, GFP- n FMRP-lele foi. Zoom (fr right), enlrgement of res outline t left. Originl mgnifition, 57 (min imges) or 4 (fr right). Dt re representtive interts with epping proteins To ientify roquin-interting proteins, we generte rt monolonl ntioy irete ginst n internl peptie of roquin (Supplementry Fig. 2,). By mss spetrometry, we ientifie the helise (n essentil omponent of the epping pthwy), the enhner of epping E4 n the epping tivtor Dp1 in extrts of T H 1 ells immunopreipitte with purifie nti-roquin ouple to mgneti es ut not in ontrol preipittions (es ouple to n irrelevnt ntioy; t not shown). We onfirme y oimmunopreipittion tht E4 n were ssoite with roquin protein in T H 1 ells (Fig. 4) n in MEFs (Fig. 4). E4 interte more strongly thn i, euse the protein seeme enrihe in immunopreipittion reltive to its presene in input. ws sustntilly n speifilly ssoite with ut ws not enrihe in nti-roquin immunopreipittions ompre with input mounts. GFP-tgge roquin(159) lso interte with enogenous E4 in nti-gfp immunopreipittes from extrts of enovirus-trnsue MEFs (Fig. 4). The intertion ourre through mino-terminl sequenes in roquin, i not require loliztion of roquin to P oies (Fig. 3 n Supplementry Fig. 1) n ws insensitive to RNse tretment uring the immunopreipittion proeure tht effiiently egre the 28S n 18S rrna in RNA extrts prepre from immunopreipittion superntnts (Fig. 4 n t not shown). Consistent with possile ssoition of ll three proteins in one omplex, we oserve full ololiztion of E4, n GFP-roquin in HEK293 ells in the sene of stress inution (Supplementry Fig. 2). Experimentlly lowere mounts of ellulr protein (Fig. 4) in MEFs trnsue with retrovirus enoing s well s retrovirus enoing roquin linke to internl riosoml entry site expression le to effetive erepression of (Fig. 4e,f). We hieve knokown y superinfetion of retrovirlly trnsue MEFs with enovirus enoing shrna speifi for the gene enoing (Dx6 (lle here); Fig. 4), n expression ws higher thn efore roquin trnsution (Fig. 4e,f). This experiment inite tht knokown of neutrlize the funtion of exogenous s well s enogenous roquin protein, whih is etetle in MEFs (t not shown). These t re onsistent with moel GFP-roquin FMRP Overly GFP-roquin FMRP Overly 2 IRES- (159) IRES 1 (WT) (1749) IRES IRES (WT) IRES in whih roquin meites silening of through funtionl n physil intertion with proteins of the epping pthwy. -meite repression oes not require mirna is not only require in the epping pthwy ut is lso essentil in mirna-epenent post-trnsriptionl silening. This pthwy hs een linke to roquin-meite repression 1. We generte MEF lones efiient in the enorionulese Dier y trnsuing ells with two loxp-flnke lleles of Dier1 (Dier1 fl/fl ), s well s wiltype ells, with retrovirus expressing Cre reominse. In prllel, we use CD4 T ells from Dier1 fl/fl n Dier1 /fl mie with Cre expression riven y C4 regultory elements. Rel-time PCR nlysis of severl nites n glol high-throughput sequening of smll RNAs showe lmost unetetle mirna in Dier-efiient MEF lones 27 (Supplementry Fig. 3,). In ontrst, lthough peripherl CD4 T ells from Dier1 fl/fl mie with C4 regultory element riven Cre expression showe effiient eletion of the trgete lleles (Supplementry Fig. 3), etetion of mirna revele more thn % of resiul enogenous expression of the mirnas mir-1, mir- 155 n mir-181 (Supplementry Fig. 3). These finings suggeste very long hlf-life of RISC-loe mirnas uring T ell evelopment n, t the sme time, rule out the possiility of testing requirement for mirna in roquin-meite expression in CD4 T ells. We therefore infete wil-type n Dier-efiient MEFs with retrovirus enoing n wil-type roquin or n roquin(159) (Fig. 5,). Wil-type roquin, unlike its intive mutnt, effiiently ownregulte surfe expression of in wil-type n Dierefiient ells (Fig. 5,). Moreover, roquin-meite repression of ourre in wil-type ells n in Dier- n mirna-efiient ells with similr ose response, s shown y quntifition of on ells with low, intermeite or high surfe expression of, the mrker oexpresse y the roquin-enoing retrovirus (Fig. 5). In ition, roquin inue similr erese of mrna expression in wil-type n Dier-efiient ells (Fig. 5). Expression of wil-type roquin n roquin(159) ws equl in wil-type n Dierefiient ells (Supplementry Fig. 3e, lne 1 versus lne 4, n lne 2 versus lne 3). We i not etet higher expression of enogenous roquin protein (Supplementry Fig. 3e, lnes 1 n 4, n f) or R3h1 mrna (t not shown) in ells with eletion of Dier. WT e expression (%) expression (%) (159) (WT) 1 (1749) (WT) of two experiments. (e) Expression of n roquin (ssesse s ) in NIH3T3 ells (,) or wil-type n TIA-1-efiient MEFs (,e) infete n ssesse s esrie ove (Fig. 2,). P =.6, roquin(1749) versus roquin(wt) (); NS, not signifint (P =.7), TIA-1-efiient versus wil-type ells (e; one-wy ANOVA). Dt re representtive of three inepenent experiments (error rs (,e), verge n s..). 2 1 Ti1 / WT NS Ti1 / 728 VOLUME 11 NUMBER 8 AUGUST nture immunology

5 Nture Ameri, In. All rights reserve. Figure 4 protein is ssoite with n E4 n shows funtionl epenene on expression. (,) Immunopreipittion (IP) of protein extrts from ulture T H 1 ells () or MEFs () with mgneti es ouple to monolonl ntiroquin or to n irrelevnt ntioy (mouse immunogloulin G (migg)); ) or isotypemthe ontrol ntioy (), followe y immunolot nlysis with ntioies (left mrgin) of immunopreipittes (right) or extrts without immunopreipittion (left). () Immunopreipittion of protein extrts, with polylonl nti-gfp, from MEFs overexpressing (overexpr) GFP-tgge eletion mutnt of roquin (roquin(159)) or GFP lone (ove lnes), with (right) or without (left) RNse tretment. EtBr (ottom) ethiium romiestine grose gel loe with RNA extrts of superntnts from immunopreipittes, to ontrol for RNAse tretment. () Immunolot nlysis of expression in MEFs trnsue with retrovirus Anti-E4 Anti- Anti-rk Anti-tuulin T H 1 extrt sh-sr We lso i funtionl tests of Dier-efiient ells n mesure mir-196-inue repression of the Hox8 3 UTR, well-estlishe trget of this mirna 28. We foun tht the Hox8 3 UTR ws onsierly represse y oexpression of onstrut tht ontine the sequene of the primry trnsript of mir-196 in wil-type ells ut not in Dier-efiient ells (Fig. 5e). In ontrst, Dier-efiient ells impire in mirna iogenesis n gene silening were still le to support roquin-meite repression of luiferse reporter through the 3 UTR of mrna (Fig. 5e). We next sought to etermine whether roquin must t together with entrl omponents of the IP m IgG sh- E4 Tuulin e Anti-E4 Anti- Anti-tuulin 2 1 MEF extrt IRES No roquin overexpr sh- IP Isotype E4 Overexpr: Anti-E4 Anti-GFP Tuulin EtBr f expression (%) GFP GFProquin(159) GFProquin(159) RNse 3 No roquin overexpr E4 GFP roquin(159) GFP roquin(159) 28S RNA 18S RNA enoing n roquin n superinfete with enovirus enoing empty vetor ontrol, srmle (sr) or -speifi shrna. (e) Expression of n roquin (ssesse s ) in ells trnsue with roquin (mile n right) or without roquin (left) n superinfete with enovirus enoing ontrol or -speifi shrna. (f) expression in the ells in e, presente reltive to expression in ontrol ells, set s %. P =.34 (one-wy ANOVA). Dt re representtive of three inepenent experiments (verge n s.. in f). Figure 5 -meite repression oes not epen on mirnas or mirisc formtion. () Expression of n roquin (ssesse s ; ) n expression of protein (,) n mrna () in Dier1 / n wil-type MEFs trnsue with retroviruses to oexpress n roquin proteins. In, expression in ells with intermeite (Int) or high (Hi) surfe expression of is ompre with tht of low (Lo) surfe expression of, set s %. In,, expression is ompre with tht of ells expressing roquin(159), set s %. P =.46 (; one-wy ANOVA). Dt re from one experiment (,) or three inepenent experiments (,; verge n s..). (e) Luiferse tivity in wil-type n Dier1 / MEFs oinfete with the ul-luiferse reporter (with Hox8 3 UTR or 3 UTR lone ehin renill luiferse) n enoviruses (left mrgin), presente s the rtio of renill luiferse units to firefly luiferse units. pri-mir-196, primry trnsript of mir-196. P <.1; NS, P =.217 (one-wy ANOVA). (f) Luiferse 2 1 mrna (%) (159) (WT) IRES IRES (WT) (159) WT Dier1 / sh- RISC to inue repression of. We teste roquin-inue ownregultion of the 3 UTR in emryoni stem ells homozygous for eletion of genes enoing the entrl RISC omponents Ago1, Ago3 n Ago4 (Eif21, Eif22 n Eif23, respetively (lle Ago1, Ago3 n Ago4 here); line B9) 29. We lso nlyze the emryoni stem ell line E7 tht is efiient in enogenous mouse Ago1Ago4 ut n still e expne in ulture euse of low expression of humn trnsgene enoing Ago2 from loxp-flnke lous. In these ells, the humn trnsgene n e lte y mens of estrogen reeptorfuse Cre reominse inue y tretment with tmoxifen. e WT Dier1 f / tivity in the emryoni stem ell lines B9 n E7 oinfete s in e n left untrete (left n mile) or trete with tmoxifen (right). P =.3, P <.1 n P <.1 (one-wy ANOVA). Dt re from three inepenent experiments (verge n s..). WT Dier1 / pri-mir-196 IRESGFP IRES-GFP IRESGFP 5 protein (%) Hox8 WT Dier1 / (WT) (159) Luiferse tivity (reltive) NS protein (%) Luiferse tivity (reltive) ES line: IRESGFP IRES-GFP Lo Int Hi B9 E7 E7 Tmoxifen WT Dier1 / nture immunology VOLUME 11 NUMBER 8 AUGUST 729

6 Nture Ameri, In. All rights reserve. Antiroquin IP Ios mrna (% of input).2.1 T H 1 extrt migg IP IP IP Mok IP mrna (% of input) (WT)GFP GFP(159) IRES- (WT) IRES (55113) IRES Figure 6 ins to mrna through its ROQ n zin-finger omins. () Immunolot nlysis of roquin in T H 1 ell extrts efore (left) n fter (right) immunopreipittion with polylonl nti-roquin es or preipittion with empty es (Mok). Dt re representtive of three experiments. () RT-PCR nlysis of enogenous Ios mrna in the preipittes in, presente s perentge of input. P =.12 (one-wy ANOVA). Dt re representtive of three inepenent experiments (verge n s..). () RT-PCR nlysis of mrna in nti-gfp immunopreipittes from extrts of HEK293 ells trnsfete with (full-length) n GFP-tgge wil-type or eletion mutnts of roquin, presente s the perentge of mrna in RNA extrts from input smples. Dt re representtive of two experiments. (,e) Expression of n roquin (ssesse s ; ) n expression (e) in MEFs sequentilly trnsue with retrovirus enoing n wil-type or mutnt roquin. P =.2; P =.1; P =.6 (one-wy ANOVA). Dt re from one experiment representtive of three () or re from three inepenent experiments (e; verge n s..). (f) Coimmunopreipittion of RNA s in. Dt re representtive of two experiments. After tmoxifen tretment, these ells remin vile for up to 4 efore unergoing poptosis 29. Aenovirl oinfetion of the 3 UTR reporter onstrut with GFP or with GFP oexpresse from n internl riosoml entry site with roquin (Fig. 5f) showe tht roquin ws le to repress the 3 UTR in ells lking mouse Ago1, Ago3 n Ago4 (line B9) n in ells lking mouse Ago1Ago4 ut ontining smll mounts of humn Ago2 (line E7). After tretment with tmoxifen, E7 ells were rreste in prolifertion ut were not impire in roquin-meite repression of, s etermine y mesurement of luiferse tivity (Fig. 5f) n flow ytometry (t not shown). The rtio of renill luiferse tivity to firefly luiferse tivity provies mesurement of 3 UTRmeite repression of renill luiferse expression. This is inepenent of infetion effiieny, epigeneti regultion of the enovirl episomes n the reltive trnsription or trnsltion effiienies of vrious ell types. Compring these normlize vlues, we i not fin eviene tht repression of the 3 UTR hnge fter prtil or omplete intivtion of mirisc funtion. Therefore, we i not etet regultion of the 3 UTR vi mirnas in emryoni stem ells (Fig. 5f). However, we nnot exlue the possiility of mirna-epenent regultion of in other ell types. In ft, we mesure mrna expression from retroviruses tht integrte into the genome of Dier-efiient n wil-type MEFs, normlize to tht of Hprt1 (enoing hypoxnthine gunine phosphoriosyl trnsferse; Supplementry Fig. 3g), n ompre tht expression with the mount of quntittive genomi PCR mplifition of the retrovirl vetorenoe GFP sequene normlize to tht of Hist1h2k (enoing histone luster 1, H2k; Supplementry Fig. 3h). We foun slightly more expression per integrtion in Dierefiient ells (Supplementry Fig. 3i), whih suggeste moerte Anti-GFP IP (55113)GFP (338113)GFP 2 1 e expression (%) Empty vetor (WT) (55113) (338113) f IP mrna (% of input) (338113) IRES Anti-GFP IP (WT)GFP GFP (1412) GFP repressive effet of mirnas on the 3 UTR in MEFs. Together these finings nnot exlue the possiility of ell typespeifi regultion of y mirnas ut rule out requirement for mirnas n RISC formtion in roquin-meite repression. is n RNA-ining protein We next ssesse whether roquin itself interts with mrna. First we resse whether we oul etet enogenous mouse Ios mrna in enogenous roquin immunopreipittes from protein extrts of primry T H 1 ells. We oumente effiient roquin protein enrihment in immunopreipittes (Fig. 6) n onfirme physil ssoition of Ios mrna n roquin protein y showing signifintly more PCR mplifition of Ios in the roquin immunopreipittion thn in mok preipittions (Fig. 6). The oserve intertion i not epen on the roxyl terminus of roquin ut epene on its mino terminus. In ft, the intertion of roquin n mrna fter otrnsfetion in HEK293 ells ws the sme or slightly greter for the roquin(159) mutnt (Fig. 6). Deletion of the mino-terminl RING finger lone in roquin(55113) h smll negtive effet on the intertion with mrna (Fig. 6). However, ining to mrna ws lost when the minoterminl eletion in the roquin(338113) mutnt inlue the ROQ omin (Fig. 6). -meite repression of ws ompletely olishe when the RING finger n ROQ omin were elete in roquin(338113), wheres eletion of the RING finger lone in roquin(55113) h prtil effet (Fig. 6,e). To ress possile ontriution of the CCCH-type zin finger, we expresse the trunte roquin protein roquin(1412), whih ontine the RING finger n ROQ omin ut lke the roxy-terminl sequenes, inluing the zin finger. This mutnt showe onsierle impirment in ining to mrna; however, it ssoite with more mrna thn ontrol immunopreipittes (Fig. 6f). These finings show tht the zin finger ts together with the ROQ omin, whih is ritil in the ining to mrna. Finlly, we investigte whether roquin, in the sene of other ellulr ftors, ws le to in n reognize mrna. We terilly expresse n purifie n mino-terminl frgment of roquin(1441), whih ontine the RING finger, ROQ n zin finger omins (Supplementry Fig. 4). We inute the protein with ppe full-length mrna generte y in vitro trnsription with T7 polymerse (Supplementry Fig. 4). In eletrophoreti moility-shift ssys, roquin protein interte with full-length mrna (Fig. 7), n the roquin-inue retrtion of the mrna n ws greter with higher onentrtions of roquin in the ining retion (Fig. 7). The progressive upshift inrese over wie rnge of protein onentrtions (Fig. 7), whih inite the existene of severl ining sites for roquin on mrna. Expression of mrna from onstrut lking the 3 UTR is not represse in ells overexpressing roquin 1. Consistent with tht fining, we i not etet ny regultion of mrna y 73 VOLUME 11 NUMBER 8 AUGUST nture immunology

7 Nture Ameri, In. All rights reserve. (1441) (p mol) Free full-length mrna Free CDS mrna Full-length mrna CDS mrna 3 (k) 6 roquin when we reple the 3 UTR of with n unrelte 3 UTR (t not shown) or with nonresponsive sequene (tht is, seon oing sequene of without strt n stop oons; Supplementry Fig. 5, ottom). Only full-length ontining the 3 UTR ws effiiently oun y roquin in vitro, wheres the oing sequene (Fig. 7) or tnem oing sequenes of (Supplementry Fig. 5) were not well reognize n their moility ws not ltere y roquin t low onentrtions (Supplementry Fig. 5). Furthermore, roquin i not lter the moility of exess yest trnsfer RNA inlue in the ining retion (Supplementry Fig. 4), n uner these onitions, n unrelte protein i not inue retrtion of the full-length mrna n (Supplementry Fig. 4). The ining ws not ue to the T7 termintor sequene (Supplementry Fig. 4e,f) n ws not epenent on the tthment of poly(a) til (t not shown). However, t high protein onentrtions, the moility of the oing sequene ws lso ltere y roquin protein. We therefore quntifie the effetive roquin protein onentrtion require for omplete ining of vrious onstruts, s etermine y the ispperne of free mrna. Full upshift of free full-length mrna eme pprent t onentrtions s low s pmol, wheres omplete upshift of oing sequene mrna i not our t onentrtions elow 1 pmol roquin per ining retion (Fig. 7), whih inite ifferene of pproximtely eightfol in the ffinity of roquin for the two ifferent mrnas. We mppe the site in the 3 UTR of require for the ining of higher ffinity y introuing progressive eletions of se pirs strting from the 3 en. An 3 UTR mrna onstrut of resiues 112, ontining the oing sequene n the first 6 se pirs of the 3 UTR, h response to roquin ining inistinguishle from tht of full-length (Supplementry Fig. 6). (1441) oun this miniml 3 UTR onstrut (112) with higher ffinity thn i n irrelevnt mrna of similr length (the oing sequene of humn FOXP3 (enoing the trnsription ftor Foxp3); Supplementry Fig. 6). By removing in stepwise Figure 7 ins to mrna with n intrinsi preferene for speifi region in the 3 UTR. (,) Eletrophoreti moilityshift ssys with inresing mounts ( pmol; ove lnes) of reominnt roquin (1441), inute with in vitrotrnsrie mrna (1 pmol) ontining the 3 UTR (full-length, 125) or lking the 3 UTR (1441) (p mol) (k) CDS (17) (1) (19) IRES (1) (CDS)(112) (118) (122) (125) (FL) Free (19) mrna Free (1) mrna Free (17) mrna Free CDS mrna (1) (oing sequene (CDS), 1; ) or with mrna in whih the 3 UTR ws progressively shortene from 9 ses ((19)) to 7 ses ((17)); ). (,) Expression of n roquin (ssesse s ; ) n () in MEFs sequentilly trnsue with retrovirus enoing roquin n or mrna with progressively shortene 3 UTR. In, expression in ells with high expression is presente reltive to its expression in -negtive ells. FL, full-length. P =.15 n P <.1 (one-wy ANOVA). Dt re from one experiment representtive of three () or re from three inepenent experiments (; verge n s..). mnner se pirs from the 3 en of this onstrut (Fig. 7), we ientifie the region etween n se pirs 3 of the stop oon s eing ritil for the higher ffinity ining of roquin to the 3 UTR. We lso orrelte the sequenes require for the higher ffinity ining of mrna y roquin with the extent of funtionl repression of y roquin in MEFs (Fig. 7,). All onstruts ontining the mppe ining site were represse y roquin. However, the egree of repression y roquin inrese with length of the 3 UTR. These finings inite tht multiple sites in the 3 UTR my tully ontriute to the funtionl repression of y roquin in living ells. DISCUSSION Our t hve inite the involvement of P-oy omponents in roquin-meite repression of. We hve shown tht loliztion of roquin to P oies ourre in primry CD4 T ells s well s in HEK293 ells n ws etete eqully y ololiztion with or E4, two ifferent P-oy mrkers. In ft, in HEK293 or CD4 T ells not sujete to stress, we i not fin ololiztion of roquin-enrihe foi with the stress grnule mrkers FMRP or G3BP1. The loliztion to P oies require the roxyl terminus of roquin, whih ontins sequenes enrihe for glutmine n sprgine. Glutmine strethes hve een shown to t s polr zippers in prion proteins 24, n more glutmine n sprgine resiues hve lso een foun in mny P-oy proteins. These regions re elieve to permit protein-protein intertions importnt for the formtion of messenger rionuleoproteins in post-trnsriptionl regultion 23,25. The presene n the funtionl importne of sequenes enrihe for glutmine n sprgine provie support for the ie of n evolutionry onserve role for roquin in the P-oy pthwy. In the ontext of the reporte loliztion of roquin to stress grnules 2,3, we note tht roquin trnslote into stress grnules fter inution of stress in HEK293 n CD4 T ells n it is therefore possile tht ells unergoing stress response hve expression (%) 1 CDS nture immunology VOLUME 11 NUMBER 8 AUGUST 731

8 Nture Ameri, In. All rights reserve. ltere roquin tivity. So fr, we hve not een le to otin experimentl support for this ie. We hve shown tht roquin effetively represse in helper T ells in phse in whih the TIA-1- epenent integrte stress response hs een shown to repress trnsltion of IL4 mrna (enoing interleukin 4) 31. However, we i not oserve ny effet on roquin tivity in MEFs efiient in TIA-1. In the P-oy pthwy, we ientifie ining of roquin to the P-oy omponents E4 n in CD4 T ells n MEFs n emonstrte the importne of expression for roquin funtion. n E4 operte in the epping pthwy n estilize mrnas in funtionl oopertion with Dp1 n Dp2, the ltter of whih removes the 5 N7-methylgunosine p. The mrnas tht lk this moifition re no longer protete from 5 -to-3 mrna egrtion 23. Nevertheless, the repression of mrna trnsltion y interferene with ining of the trnsltion-initition ftor omplex eif4f to the 5 N7-methylgunosine p of the mrna represents the rte-limiting step of epping Therefore, lthough they o not exlue the possiility of role for roquin in the first step of trnsltionl repression, our results suggest tht roquin promotes epping of trget mrnas. We hve lso presente eviene tht roquin is n RNA-ining protein. The oserve ssoition of roquin n mrna in T ell extrts or ell extrts from trnsfete HEK293 ells epene minly on the ROQ omin, whih shows tht this omin is memer of reltively smll group of RNA-ining moules 35. It seems tht the ROQ omin ins in omintion with the CCCH-type zin finger moule in roquin. This moe of protein-rna intertion llowe ining of multiple roquin moleules to the sme mrna in vitro. The oopertion of ifferent RNA-ining moules in one protein hs een esrie s ommon feture of RNA-ining proteins n typilly enles them to in with higher ffinity or to otin speifiity for n mrna sustrte mong n enormous iversity of possile strutures 35. These finings re onsistent with report lso emonstrting tht the ROQ omin is ritil for the ining of roquin to short RNA frgment from the 3 UTR 3. However, we foun tht roquin h preferene for ertin struture or sequene eterminnts present in previously unreognize region of the mrna, whih proly speify the ellulr trgets of roquin. Our experiments showe roquin oun with higher ffinity to speifi sequene lote etween n se pirs ownstrem of the stop oon in the 3 UTR. In ition, we foun generl ining to vrious other mrnas with lower ffinity, whih inites the possiility tht sequentil ining on the mrna oul our in oopertive mnner. We onlue tht roquin is protein tht hs low ffinity for mrnas in generl. However, roquin hs lso protein-intrinsi higher ffinity for sequene or struture eterminnts of the 3 UTR. The ft tht the 3 UTR ontriutes more regultory elements thn just one single roquin-speifi ining site for effiient repression of in ells inites the involvement of itionl steps eyon ining. Therefore, we propose tht the repression ours in higher orer struture inue y reognition of the 3 UTR y roquin ut t the sme time nees ellulr oftors tht my require itionl fetures in the 3 UTR. We nlyze roquin tivity in ells efiient in Dier n Ago1 Ago4. We i not fin eviene for positive role of the mirisc, s suggeste y stuies esriing tht silening of y roquin requires n intt mir-1-reognition motif 1. In those experiments, ritil region in the 3 UTR of ws mppe n miniml response element of 47 se pirs ws efine 1. We ssume tht experimentl ifferenes relte to the use of isolte frgments linke to reporters or, in our se, the entire 3 UTR in the ontext of the oing sequene my ount for the ontrsting onlusions. The newly esrie moleulr funtions of roquin my e involve in the prevention of utoimmunity. These funtions inlue sequeneor struture-speifi ining to RNA s well s the formtion of omplexes with other ftors involve in mrna ey. We speulte tht t lest one of these funtions oul e impire in roquin proteins with the methionine-to-rginine sustitution t position 199 in the ROQ omin n oul therey use the evelopment of utoimmune isese in R3h1 sn/sn mie 2. Methos Methos n ny ssoite referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Immunology wesite. Aknowlegments We thnk M. Shmit-Supprin n D. Niessing for ritil reing of the mnusript n isussions; G. Stoeklin for unpulishe oservtions n vie; C. Vinues (The John Curtin Shool of Meil Reserh, Cnerr, Austrli) for n roquin DNA; G. Hnnon (Col Spring Hror Lortory) for Dier1 fl/ mie; C. Wilson (University of Wshington) for Tg(C4-re) mie; N. Keersh n P. Anerson (Brighm n Women s Hospitl) for TIA-1-knokout MEFs; U. Fisher (Biozentrum Würzurg) for nti-fmrp; H. Srioglu for mss-spetrometry nlysis; n C. Thirion n L. Behren (Sirion Bioteh) for vie n regents for enovirl infetion. Supporte y Deutshe Forshungsgemeinshft (SFB 571 to V.H.) n Fritz Thyssen Fountion (V.H.). AUTHOR CONTRIBUTIONS E.G. i most experiments, with the help of K.P.H., N.R. n C.W.; K.U.V. ontriute to some experiments n eite the mnusript; L.D., E.K. n X.W. estlishe tools n provie vie; E.G. n V.H. plnne the projet together; n V.H. supervise the experiments n wrote the mnusript. COMPETING FINANCIAL INTERESTS The uthors elre no ompeting finnil interests. Pulishe online t Reprints n permissions informtion is ville online t reprintsnpermissions/. 1. Yu, D. et l. represses utoimmunity y limiting inuile T-ell o-stimultor messenger RNA. Nture 45, (7). 2. Vinues, C.G. et l. A RING-type uiquitin ligse fmily memer require to repress folliulr helper T ells n utoimmunity. Nture 435, (5). 3. Anerson, P., Phillips, K., Stoeklin, G. & Keersh, N. Post-trnsriptionl regultion of proinflmmtory proteins. J. Leuko. Biol. 76, 4247 (4). 4. Ho, S. & Bltimore, D. The stility of mrna influenes the temporl orer of the inution of genes enoing inflmmtory moleules. Nt. Immunol., (9). 5. Hoefig, K.P. & Heissmeyer, V. MiroRNAs grow up in the immune system. Curr. Opin. Immunol., (8). 6. Stefl, R., Skrisovsk, L. & Allin, F.H. RNA sequene- n shpe-epenent reognition y proteins in the rionuleoprotein prtile. EMBO Rep. 6, 3338 (5). 7. Jing, Q. et l. Involvement of mirorna in AU-rih element-meite mrna instility. Cell 1, (5). 8. Kee, M. et l. RNA-ining protein Dn1 inhiits mirorna ess to trget mrna. Cell 131, (7). 9. Vsuevn, S. & Steitz, J.A. AU-rih-element-meite upregultion of trnsltion y FXR1 n Argonute 2. Cell 128, (7).. Tn, A.H., Wong, S.C. & Lm, K.P. Regultion of mouse inuile ostimultor () expression y Fyn-NFAT2 n ERK signling in T ells. J. Biol. Chem. 281, (6). 11. Bossller, L. et l. efiieny is ssoite with severe reution of CXCR5CD4 germinl enter Th ells. J. Immunol. 177, (6). 12. Dong, C. et l. o-stimultory reeptor is essentil for T-ell tivtion n funtion. Nture 9, 971 (1). 13. Dong, C., Temnn, U.A. & Flvell, R.A. Cutting ege: ritil role of inuile ostimultor in germinl enter retions. J. Immunol. 166, (1). 732 VOLUME 11 NUMBER 8 AUGUST nture immunology

9 14. MAm, A.J. et l. Mouse inuile ostimultory moleule () expression is enhne y CD28 ostimultion n regultes ifferentition of CD4 T ells. J. Immunol. 165, 5355 (). 15. Tfuri, A. et l. is essentil for effetive T-helper-ell responses. Nture 9, 59 (1). 16. Lintermn, M.A. et l. Folliulr helper T ells re require for systemi utoimmunity. J. Exp. Me. 6, (9). 17. Wn, Y.Y. et l. Trnsgeni expression of the oxskie/enovirus reeptor enles enovirl-meite gene elivery in nive T ells. Pro. Ntl. A. Si. USA 97, (). 18. Oeroerffer, P. et l. Effiieny of RNA interferene in the mouse hemtopoieti system vries etween ell types n evelopmentl stges. Mol. Cell. Biol. 25, (5). 19. Heissmeyer, V., Ansel, K.M. & Ro, A. A plgue of utontioies. Nt. Immunol. 6, (5).. Chu, C.Y. & Rn, T.M. Trnsltion repression in humn ells y mirorna-inue gene silening requires RCK/p54. PLoS Biol. 4, e2 (6). 21. Diiot, M.C., Surmnin, M., Fltter, E., Mnel, J.L. & Moine, H. Cells lking the frgile X mentl retrtion protein (FMRP) hve norml RISC tivity ut exhiit ltere stress grnule ssemly. Mol. Biol. Cell, (9). 22. Mzroui, R. et l. Trpping of messenger RNA y frgile X mentl retrtion protein into ytoplsmi grnules inues trnsltion repression. Hum. Mol. Genet. 11, (2). 23. Frnks, T.M. & Lykke-Anersen, J. The ontrol of mrna epping n P-oy formtion. Mol. Cell 32, 5615 (8). 24. Mihelitsh, M.D. & Weissmn, J.S. A ensus of glutmine/sprgine-rih regions: implitions for their onserve funtion n the preition of novel prions. Pro. Ntl. A. Si. USA 97, (). 25. Reijns, M.A., Alexner, R.D., Spiller, M.P. & Beggs, J.D. A role for Q/N-rih ggregtionprone regions in P-oy loliztion. J. Cell Si. 121, (8). 26. Gilks, N. et l. Stress grnule ssemly is meite y prion-like ggregtion of TIA-1. Mol. Biol. Cell 15, (4). 27. Prmeswrn, P. et l. Six RNA viruses n forty-one hosts: virl smll RNAs n moultion of smll RNA repertoires in verterte n inverterte systems. PLoS Pthog. 6, e764 (). 28. Yekt, S., Tin, C.J. & Brtel, D.P. MiroRNAs in the Hox network: n pprent link to posterior prevlene. Nt. Rev. Genet. 9, (8). 29. Su, H., Tromly, M.I., Chen, J. & Wng, X. Essentil n overlpping funtions for mmmlin Argonutes in mirorna silening. Genes Dev. 23, (9). 3. Athnsopoulos, V. et l. The ROQUIN fmily of proteins lolizes to stress grnules vi the ROQ omin n ins trget mrnas. FEBS J. 277, (). 31. Sheu, S. et l. Ativtion of the integrte stress response uring T helper ell ifferentition. Nt. Immunol. 7, (6). 32. Cougot, N., vn Dijk, E., Bjko, S. & Serphin, B. Cp-tolism. Trens Biohem. Si. 29, (4). 33. Eullio, A., Behm-Ansmnt, I. & Izurrle, E. P oies: t the rossros of posttrnsriptionl pthwys. Nt. Rev. Mol. Cell Biol. 8, 922 (7). 34. Prker, R. & Sheth, U. P oies n the ontrol of mrna trnsltion n egrtion. Mol. Cell 25, (7). 35. Lune, B.M., Moore, C. & Vrni, G. RNA-ining proteins: moulr esign for effiient funtion. Nt. Rev. Mol. Cell Biol. 8, (7). Nture Ameri, In. All rights reserve. nture immunology VOLUME 11 NUMBER 8 AUGUST 733

10 Nture Ameri, In. All rights reserve. ONLINE METHODS Mie. Mie (from Toni Frms) were house in speifi pthogenfree rrier fility n were use t 612 weeks of ge in orne with the Helmholtz Zentrum Münhen institutionl, stte n feerl guielines. Cell ulture n trnsfetion. HEK293 ells n MEFs were grown in DMEM with % (vol/vol) FCS, peniillin-streptomyin (1, U/ml) n HEPES ( mm), ph 7.4. Emryoni stem ells were grown first on feeer ells in DMEM ontining % (vol/vol) FCS, L-glutmine (2 mm), peniillinstreptomyin ( U/ml), soium pyruvte (1 mm), nonessentil mino is (.1 mm), β-merptoethnol (.1 mm) n leukemi-inhiitory ftor (2, U/ml) n were pssge twie on.1% (wt/vol) geltin. Peripherl CD4 T ells were isolte with CD4 Dynes (Invitrogen), were kept in RPMI meium (with % (vol/vol) FCS, β-merptoethnol (.1 mm), peniillin-streptomyin ( U/ml) n HEPES ( mm), ph 7.2) n were stimulte for 3648 h uner T H onitions (on surfes ote with got ntihmster immunogloulin G (55397; MP Biomeils) plus nti-cd3 (.1 μg/ml; 145-2C11) n nti-cd28 (1 μg/ml; 37N); oth purifie in-house); T H 1 onitions (inlusion of ntioy to interleukin 4 (nti-il-4; μg/ml; IIB11; purifie in-house) n reominnt IL-12 ( ng/ml; R&D Systems); or T H 2 onitions (inlusion of nti-il-12 (3 μg/ml; C17.8) n nti-interferon-γ (5 μg/ml; Xmg1.2); oth purifie in-house, s well s superntnts of I3L6 mouse myelom ells ontining mouse IL-4 (whih orrespons to 1, U/ml of reominnt IL-4)). Tosyl-tivte M-45 es (Invitrogen) ouple to nti-cd28 n nti-cd3 were use for e stimultion. After eing stimulte for 48 h, ell popultions were expne in reominnt humn IL-2 ( U/ml; Worl Helth Orgniztion, Ntionl Institute for Biologil Stnrs n Control). All monolonl ntioies were purifie from superntnts of hyrioms on protein A Sephrose olumns (145-2C11 n 37N) or protein G Sephrose olumns (Xmg1.2, IIB11, C17.8 n nti-roq hyrioms) n were ilyze ginst PBS. Virus proution. Type 5 replition-efiient enovirus ws proue y trnsfetion of enovirl vetors into HEK293 ells, followe y purifition oring to the mnufturer s instrutions (Cell Biols). Retrovirl superntnts were proue y lium-phosphte trnsfetion of HEK293T ells with mphotropi pkging vetors n retrovirl expression vetors. Superntnts were ollete 72 h fter trnsfetion, were filtere through.45-μm filters n were use for spin-infetion. Trnsue ells were nlyze 34 fter infetion on FACSCliur or were sorte on FACSAri II (Beton Dikinson). Aenovirl knokown or etopi protein expression. Aenovirl vetors (Sirion Bioteh) with ontrol shrna (5 -TTTTTGGCCTTTTTTAGCTG-3 ), srmle shrna (5 -ACAAGATGAAGAGCACCAA-3 ), R3h1-speifi shrna (5 -CGCACAGTTACAGAGCTCATT-3 ), C4-speifi shrna (5 -CACAGCTATCACGGCCTATAA-3 ) or -speifi shrna (5 -GCC AAGAACTATGTCTTTATA-3 ) were ple uner the ontrol of polymerse IIIepenent smll nuler RNA U6 promoter n were oexpresse with phosphoglyerte kinse promoterriven GFP. The enovirl expression vetor ws rete y ssemly of the rtifiil CAG promoter in front of Gtewy ssette followe y poly(a) signl from ovine growth hormone in the pa-pl vetor (Invitrogen), into whih GFP-tgge roquin ws inserte y mens of lm reomintion from the pentr 11 vetor (Invitrogen). mirna ws mesure y quntittive PCR n normlize to tht of the smll nuler RNA U6 fter the use of TqMn mirorna Reverse Trnsription kit n stem-loop primers speifi for mir-1, mir-328, mir-196 mir-214, mir-155, mir-181 n U6 (Applie Biosystems). A LightCyler 4II n Light Cyler 4 softwre relese 1.5. SP1 were use for quntittive PCR. Confol mirosopy. HEK293 ells were trnsfete with FuGENE regent (Rohe), then were seee on glss over slips n sujete to stress for 1 h t 37 C with.5 mm soium rsenite (Sigm). Dignosti glss over slips were ote first with.1% (wt/vol) poly-l-lysine n then with nti- CD3 (2.5 μg/ml) n nti-cd28 (5 μg/ml) efore the ition of T ells in volume of 5 μl. Cells were fixe with 4% (wt/vol) prformlehye n were wshe three times in.5% (vol/vol) Noniet-P,.1% (wt/vol) NN 3 n % (vol/vol) FCS in PBS. Antioy to p7 S6 kinse-α (s-8418; Snt Cruz), nti-g3bp (s-819; Snt Cruz) n nti- (A3-461A; Bethyl) or nti-fmrp ( gift from U. Fisher) ws use for stining in omintion with seonry ntioies ouple to inoiroynine ( ; Jkson Lortories) or inoroynine ( ; Jkson Lortories). Imges were pture on Lei DM IRBE mirosope n were nlyze with LCS Lite softwre. Coimmunopreipittion of roquin-ssoite mrna or protein. This ws one s esrie 36. T ells (2 8 ) or HEK293 ells (1 7 ) were lyse on ie in 2 ml lysis uffer ( mm Tris, ph 7.5, 15 mm NCl,.25% (vol/vol) Noniet-P, 1.5 mm MgCl 2, protese inhiitor mix without EDTA (Rohe) n 1 mm ithiothreitol). Lystes were pssge through 26-guge neele, then were shok frozen n thwe n then lere y entrifugtion. Antiroquin (A3-514A; Bethyl) or nti-gfp (A11122; Invitrogen) oun to protein G mgneti es (Invitrogen) ws inute for 4 h t 4 C with lystes in the presene of U RNsin. Bes were wshe two times (RNA) or three times (protein) with lysis uffer with the inlusion of two itionl wshes (RNA) with lysis uffer ontining 3 mm NCl,.5% (vol/vol) Noniet-P n 2.5 mm MgCl 2. Purifition of roquin protein. (1441) ws expresse from the petm11 teril expression vetor (moifie; Novgen) in teri, ws purifie y histiine-tg hromtogrphy (GE Helthre) n ws eslte vi PD gel filtrtion (GE Helthre). In vitro RNA trnsription. For in vitro trnsription, inserts ontining the mrna n 3 UTR in the teril expression vetor pdest17 (Invitrogen) were linerize y igestion with restrition enzymes. The mmessge mmhine T7 trnsription kit ws use oring to the mnufturers instrutions (Amion) for in vitro trnsription of mrna, n RNA ws purifie with n RNesy Kit (Qigen). Mss spetrometry. For the ientifition of oimmunopreipitte proteins, slies of SDS-polyrylmie gels were nlyze y liqui hromtogrphy tnem mss spetrometry pproh with n LTQ Oritrp XL mss spetrometer ouple to n Ultimte 3 Nno-HPLC. All tnem mss spetrometry spetr were then nlyze with the Msot serh engine ginst the UniProt referene luster (uniref_mouse fst) protein tse. Protein ientifitions were epte if they oul e estlishe t proility of greter thn 95% n ontine t lest two ientifie pepties. Luiferse ssy. MEFs plte on 12-well pltes were infete with enovirus. After 2, ul-luiferse reporter ssy (Promeg) ws one. Cells were lyse in 15 μl lysis uffer, lystes were lere y entrifugtion n μl superntnt ws nlyze. Rel-time PCR. RNA ws isolte from ells or immunopreipittes with TRIzol (Sigm) or MirVn kit (Amion), respetively. After reverse trnsription with the Quntitet kit (Qigen,) quntittive PCR ssys were one (primers n universl proes, Supplementry Tle 1; Rohe). Expression of Eletrophoreti moility-shift ssy. Smples were seprte y eletrophoresis through ntive.75% grose gels, then were stine with ethiium romie. Reominnt protein (51 pmol) ws inute for min on ie with mrna (.6 or 1 pmol) in solution of 15 mm KCl, 5 mm Tris- HCl, ph 7.4, 1 mm MgCl 2, 1 mm EDTA n 1 mm ithiothreitol, n then ws juste to onentrtion of 16% (vol/vol) glyerol. 36. Weinmnn, L. et l. Importin 8 is gene silening ftor tht trgets rgonute proteins to istint mrnas. Cell 136, (9). nture immunology oi:.38/ni.192