MicroRNA-dependent localization of targeted mrnas to mammalian P-bodies

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1 MicroRNA-dependent locliztion of trgeted mrnas to mmmlin P-odies Jidong Liu 1, Mrco Antonio Vlenci-Snchez 2, Gregory J. Hnnon 1,3 nd Roy Prker 2,3 Smll RNAs, including smll interfering RNAs (sirnas) nd micrornas (mirnas) cn silence trget genes through severl different effector mechnisms 1. Wheres sirna-directed mrna clevge is incresingly understood, the mechnisms y which mirnas repress protein synthesis re oscure. Recent studies hve reveled the existence of specific cytoplsmic foci, referred to herein s processing odies (P-odies), which contin untrnslted mrnas nd cn serve s sites of mrna degrdtion 2 7. Here we demonstrte tht Argonute proteins the signture components of the RNA interference (RNAi) effector complex, RISC loclize to mmmlin P-odies. Moreover, reporter mrnas tht re trgeted for trnsltionl repression y endogenous or exogenous mirnas ecome concentrted in P-odies in mirna-dependent mnner. These results provide link etween mirna function nd mmmlin P-odies nd suggest tht trnsltion repression y RISC delivers mrnas to P-odies, either s cuse or s consequence of inhiiting protein synthesis. RNAi ws initilly chrcterized s post-trnscriptionl gene silencing mechnism in which the experimentl introduction of long doule-strnded RNAs (dsrnas) induces sequence-specific destruction of homologous mrnas (reviewed in ref. 1). RNAi pthwys re initited when dsrnas re processed y Dicer into sirnas of nucleotides. sirnas re incorported into the effector complex RISC. In RISC, the sirna is ound y n Argonute protein, which uses the sequence of the sirna to select nd cleve complementry sustrtes (reviewed in ref. 8). RISC cn lso silence gene expression y preventing protein synthesis. Genetic studies of Cenorhditis elegns tht re mutnt for Dicer forged the initil link etween previously known clss of smll regultory RNAs, the strnas, nd the RNAi pthwy Susequent studies showed tht strnas re rchetypes of lrge clss of regultory RNAs, known s mirnas (reviewed in ref. 14). Although mirna nd sirna pthwys cn e iochemiclly comprtmentlized, oth types of RNAs enter RISC, ind to Argonute proteins nd identify their silencing trgets in conceptully similr wys. They differ, t lest in nimls, in tht mirnas most often pir imperfectly with their trgets nd re thus unle to direct Argonute-medited clevge 15. Insted, mirnas repress protein synthesis in clevge-independent fshion 8,14,15. The mechnism y which mirnas repress trnsltion of their trget mrnas is unknown. Conceivly, RISC could prevent protein synthesis from mirna trgets in one of severl wys. RISC could ffect trnsltion, per se, y ltering rtes of initition or elongtion y the riosome. Alterntively, trnsltion could proceed unffected with nscent polypeptides eing degrded. A third possiility is tht trget mrnas could e somehow sequestered from the trnsltionl mchinery. To investigte this third model, we ssessed the locliztion of n ectopiclly expressed, -tgged humn Argonute protein,, which is fully functionl for sirna-medited silencing 16. Immunofluorescence indicted pttern of locliztion in discrete cytoplsmic foci, lthough some my lso e distriuted throughout the cytoplsm (Fig. 1). Both the size of individul foci nd the numer of foci vried etween individul cells with n verge of four to nine cler foci per cell. Endogenous could lso e detected in foci with similr verge numer of three to six foci per cell (Fig. 1). Notly, these foci were oserved when the ntiody ws used to stin wild-type ut not -mutnt mouse emryo firolsts 16, indicting tht this pttern truly reflects the locliztion of the endogenous protein (dt not shown). Differences in numers of pprent foci proly result from nti- ntiodies giving overll weker signls thn did monoclonls tht recognize epitope-tgged proteins. ppered in foci irrespective of its ility to cleve trget RNAs, s demonstrted y the locliztion of ctlyticlly incompetent mutnts (see Supplementry Informtion, Fig. S1). Similr locliztion ptterns were lso seen for ll other mmmlin Argonute proteins tht hve een shown to ind mirnas (, -3 nd -4; see Supplementry Informtion, Fig. S2). In contrst, memers of the second Argonute sufmily, the Piwi fmily, hve not een shown to interct with mirnas nd do not loclize to cytoplsmic foci when ectopiclly expressed (for exmple, Hiwi; dt not shown). The pttern nd size of the Argonute-contining foci were reminiscent of the cytoplsmic P-odies (lso termed Dcp odies or GW odies) tht hve previously een oserved in oth yest nd niml cells 2 5,7,17. To test the possiility tht Argonute-contining foci were P-odies, we 1 Cold Spring Hror Lortory, Wtson School of Biologicl Sciences, 1 Bungtown Rod, Cold Spring Hror, NY 11724, USA. 2 Deprtment of Moleculr nd Cellulr Biology & Howrd Hughes Medicl Institute, University of Arizon, Tucson, AZ 85721, USA. 3 Correspondence should e ddressed to G.J.H. (e-mil: hnnon@cshl.org) nd R.P. (e-mil: rrprker@u.rizon.edu) Pulished online: 5 June 2005; DOI: /nc1274 NATURE CELL BIOLOGY VOLUME 7 NUMBER 7 JULY

2 c / colocliztion Flg GFP / colocliztion Flg Endogenous GFP Figure 1 Argonute proteins loclize to mmmlin P-odies. () -tgged protein ws expressed in U2-OS cells. protein loclized to discrete cytoplsmic foci y stining with FITC-conjugted nti-. () Endogenous protein ws loclized in U2-OS cells y stining with rit nti- ntiody. (c) Argonute proteins coloclized with either GFP- or Flg-tgged, signture component of the mmmlin P-odies. Argonute proteins were visulized using Rhodmine-Red-conjugted nti-. ws visulized either y GFP or FITC-conjugted nti-flg. sked whether Ago proteins coloclized with known P-ody components. is component of the decpping enzyme nd hs een demonstrted to reside in P-odies in oth yest nd mmmls 2 5,7. A comprison of the locliztion pttern of or with tht of either Flg epitope- or green fluorescent protein (GFP)-tgged showed remrkle overlp (Fig. 1c). In some cses, the reltive intensity of the nd Ago proteins in n individul focus vried, ut close inspection revels tht essentilly ll nd Ago foci overlp. Becuse Argonute proteins were loclized to P-odies, we sked whether we could oserve iochemicl interction etween Ago nd other P-ody components. We could esily detect the presence of either GFP- or Flg-tgged in immunoprecipittes of -tgged or (Fig. 2). Moreover, when GFP ws immunoprecipitted with GFP ntiody, nd were co-immunoprecipitted (Fig. 2). Interction ws lso oserved etween / nd second suunit of the decpping enzyme, (Fig. 2). The physicl interction etween Argonutes nd or proteins could e protein protein interction, or could e the result of these proteins intercting with common mrna or P-ody structure. To distinguish etween these possiilities, we treted lystes, prior to immunoprecipittion, with RNseA, which oth degrdes mrnas nd destroys P-ody integrity 6,18. Even fter such tretment, the interction etween nd GFP or Flg ws preserved (Fig. 2c, d). These results indicte tht mmmlin Ago sufmily proteins re not only concentrted in P-odies, ut lso interct physiclly with the P- ody components nd in mnner tht is independent of rionucleoprotein (RNP) or P-ody integrity. The Argonute proteins could ccumulte in P-odies ecuse of protein protein interctions, independent of their ility to function in RNAi. Alterntively, the Argonute proteins could e trgeted to P-odies in mnner tht depends upon intct interctions with smll RNAs or even upon successful mirna mrna recognition. We generted two mutnt proteins using the crystl structure of the humn PAZ domin ound to n sirna-like duplex s guide 19. These mutnts, -PAZ9 nd -PAZ10, contin either nine or ten point muttions within the PAZ domin of. Both mutnts show sustntilly reduced ility to interct with smll RNAs or to cleve trget mrnas (Fig. 3). Notly, oth mutnts filed to ccumulte in P-odies (Fig. 3). These results re consistent with the notion tht Argonute requires interction with smll RNA for its ccumultion in P-odies. This outcome could suggest specific recognition of Ago proteins loded with mirna/sirnas, either with or without trget mrnas, y fctor tht medites P-ody locliztion. Alterntively, the muttions could hve mrkedly ltered the overll structure of the proteins such tht they were completely non-functionl. Contrry to the ltter possiility, oth -PAZ9 nd -PAZ10 mutnts were expressed t norml levels nd retined the ility to interct with nd (Fig. 3, c). This ltter oservtion rgues tht the Ago or Ago interctions re not sufficient to recruit Ago proteins to the 720 NATURE CELL BIOLOGY VOLUME 7 NUMBER 7 JULY 2005

3 Trnsfection Anti-GFP Trnsfection GFP Anti-Flg IP Anti-GFP Flg IP Anti-Flg GFP RNseA + + IP Anti-GFP d Flg RNseA + + IP Anti-Flg / Anti-GFP / Anti-Flg Figure 2 Argonute proteins ind components of mmmlin P-odies. Humn 293T cells were trnsfected with -tgged or in comintion with either GFP-tgged or Flg-tgged expression plsmids (s indicted). () or (nti-) or (nti-gfp) immunoprecipittes were western lotted with nti- or nti-gfp ntiodies s indicted. () or (nti-) or (nti-flg) immunoprecipittes were western lotted with nti- or nti-flg ntiodies s indicted. (c) Extrcts were treted with RNseA efore immunoprecipittion of (nti-) or (nti-gfp). Immunocomplexes were western lotted with nti- or nti-gfp ntiodies s indicted. (d) Extrcts were treted with RNseA efore immunoprecipittion of (nti-) or (nti-flg). Immunocomplexes were western lotted with nti- or nti-flg ntiodies s indicted. mmmlin P-odies. Thus, the simplest interprettion of the filure of -PAZ9 or -PAZ10 to loclize to P-odies is tht mirna inding to is required for its ccumultion within P-odies. The presence of Argonute proteins within P-odies suggests tht mirna-medited repression of protein synthesis might result in the trgeting of mirna mrna complexes to P-odies. To test this hypothesis, we sked whether n mrna trgeted for trnsltionl repression y mirna would ccumulte within P-odies. We generted luciferse mrna expression constructs with nd without portion of the C. elegns lin-41 3 UTR, which is trget of the let-7 mirna. Both the let-7 trget nd its control counterprt lso contined 24 inding sites for the MS2 cot protein in their 3 UTRs (Fig. 4, ). These MS2-inding sites llowed us to follow the locliztion of these mrnas y co-expression of n MS2 YFP NLS fusion protein. In cells tht lck trget mrna, the fusion protein remins loclized to the nucleus, reducing ckground cytoplsmic fluorescence. However, in the presence of n mrna contining pproprite inding sites, frction of the fusion protein is crried into the cytoplsm, where its locliztion reports the loction of the trget mrna 20. Expression of the let-7 trget in U2-OS cells, which endogenously express undnt endogenous let-7 mirna, provided two oservtions. First, the construct contining the lin-41 3 UTR frgment genertes ~twofold less luciferse thn the control trnscript tht does not contin the sites. c c Trnsfection GFP Anti-GFP Wild-type -PAZ9 -PAZ10 Wild-type -PAZ9 -PAZ10 sirna lot Slicer ssy Wild-type -PAZ9 -PAZ10 IP Anti-GFP Both the site-dependence nd the mgnitude of the chnge in the reporter re consistent with previously oserved regultion of similr reporters y let- 7 (ref. 21). A second, nd criticl, oservtion ws tht when the let-7 trget ws expressed in U2-OS cells, we oserved discrete cytoplsmic foci of the MS2 YFP NLS fusion protein tht coloclized with (Fig. 4). Colocliztion ws not unique to ut ws lso oserved for other Argonute sufmily memers (see Supplementry Informtion, Fig. S3). This oservtion indicted tht the let-7 trget mrna concentrted in cytoplsmic P-odies. Notly, expression of trget mrna tht lcked the let-7 inding sites showed no cytoplsmic foci of the MS2 YFP NLS protein, indicting tht this mrna ws not concentrted in P-odies (Fig. 4). The forementioned dt indicte tht n mrna cn e loclized to P-odies in mnner tht is dependent on the presence of mirna-inding site. However, ecuse let-7 is endogenously expressed, we could not ensure tht locliztion ws mirna directed. Thus, we Wild-type -PAZ9 -PAZ10 -PAZ10 Figure 3 Accumultion of Argonute proteins in P-odies requires n intct sirna-inding domin. () Wild-type nd mutnt proteins, -PAZ9 nd -PAZ10 (s indicted), were expressed s epitope fusions in cells trnsfected with luciferse sirna. ting with n nti- ntiody ws used to mesure protein expression (upper pnel). Smll RNA inding ws mesured y northern lotting of RNA extrcted from immunocomplexes (middle pnel). Nuclese ctivity ws lso mesured ginst complementry sustrte (ottom pnel). () Sucellulr locliztion of wild-type nd non-smll RNA inding mutnt proteins ws determined y immunostining with nti- ntiodies. (c) -tgged wild-type or mutnt proteins were co-expressed with GFP-tgged. (nti-) or (nti-gfp) immunoprecipittes were nlysed y western lotting with nti- or nti-gfp ntiodies, s indicted. NATURE CELL BIOLOGY VOLUME 7 NUMBER 7 JULY

4 YFP MS2 MS2 YFP c YFP MS2 MS2 YFP MS2 YFP NLS MS2 YFP NLS let-7 mirna d YFPMS2 MS2 YFP Exogenous CXCR4 sirna MS2 YFP NLS YFP MS2 MS2 YFP MS2 YFP NLS Endogenous MS2 YFP NLS Figure 4 mirna-dependent locliztion of trget mrnas to mmmlin P-odies. () Plsmids expressing the let-7 trget, protein nd MS2 YFP NLS were cotrnsfected into U2-OS cells. Reporter mrna ws visulized indirectly using the fusion protein. protein ws visulized using Rhodmine-Red-conjugted nti-. () Anlyses were identicl to in except tht the trget mrna did not contin the lin-41 3 UTR frgment. constructed trget tht contins oth the MS2-inding region nd multiple imperfect complements of CXCR4 sirna tht hs een shown to ct s mirna mimetic, repressing trget expression in the sence of slicer clevge 22 (Fig. 4c, d). Co-expression of the CXCR4 trget with the fluorescent MS2 protein in the sence of the CXCR4 sirna showed no cler foci (Fig. 4c). However, when the sirna ws co-delivered, we oserved tht the CXCR4 trget ws concentrted in cytoplsmic foci tht coloclized with, Ago3 nd endogenous proteins (Fig. 4d nd see Supplementry Informtion, Fig. S3). Concomitnt with its locliztion in P-odies, the expression of the reporter ws reduced y two- to threefold (dt not shown). Considered together, our results indicte tht mirna-regulted mrnas cn ccumulte in mmmlin P-odies in mnner tht depends oth on the presence of the smll RNA nd upon the presence of the pproprite recognition sites in the trget mrna. Severl oservtions in this work now indicte connection etween mmmlin P-odies nd mirna-regulted repression of mrnas. First, Argonute proteins loclize to P-odies. Second, locliztion of to P-odies requires the ility to ind mirnas. Third, two different reporter mrnas one trget of the endogenous let-7 mirna nd one trget of n rtificil mirna mimic ccumulte in P-odies in (c) The CXCR4 trget ws co-expressed with MS2 YFP NLS nd. Detection ws s in. (d) The CXCR4 reporter ws co-expressed with MS2 YFP NLS nd with (upper) or without (lower) in cells tht were lso trnsfected with the CXCR4 sirna. Endogenous ws visulized with rit nti- ntiody. Digrmmtic representtion trgets re shown next to ech pnel. mnner tht is dependent on the mirna, or the mirna-inding sites. Fourth, nd co-immunoprecipitte with the P-ody components, nd. Moreover, ecuse Dcp Ago interctions re resistnt to RNseA nd occur with mutnts tht do not loclize to P-odies, this interction is likely to occur oth within nd outside of P-odies nd could hve n erly role in the trgeting of mirna trgets to P-odies. Considered together, strightforwrd hypothesis sed on these oservtions is tht mrnas undergoing mirna-medited trnsltion repression ccumulte within P-odies in conjunction with n Argonute mirna complex. Our results re consistent with severl previous studies tht suggest connection etween P-odies nd the RNAi mchinery. Homologues of Xrn1p, 5 to 3 exonuclese tht is concentrted in P-odies, hve een shown in plnts nd in Drosophil melnogster S2 cells to degrde the 3 products tht rise from smll RNA-directed clevge of mrnas 23,24. Strikingly, Xrn1p is required for efficient RNAi in C. elegns 25. Additionlly, muttion of Xrn4 in Aridopsis thlin induced n RNAi response ginst certin trnsgenes, presumly ecuse of the ccumultion of errnt RNAs 26. Our results my lso provide n explntion for slicerindependent reduction in the undnce of proposed mirna trgets in 722 NATURE CELL BIOLOGY VOLUME 7 NUMBER 7 JULY 2005

5 mmmls 27. Sequestrtion of trnsltionlly repressed mrnas to P-odies nd the direct interction etween Argonutes nd the Dcp1/ complex might promote decpping of trgets, therey enhncing their degrdtion, which might lso explin the mirna-dependent increse in mrna decy seen for AU rich element (ARE)-contining mrnas 28. An issue tht remins to e resolved is the precise nture of the functionl connection etween mirna-medited regultion nd P-odies. In one model, mirnas presumly in conjunction with Argonute proteins could repress the trnsltion of their trget genes y ffecting some spect of trnsltion, per se. As consequence, the trgeted mrnas could e recognized y n unknown mchinery nd tken to the P-ody for sequestrtion nd perhps ultimte disposl. In this regrd, it should e noted tht, t lest in yest, defects in trnsltion elongtion prevent mrnas from entering P-odies, nd defects in trnsltion initition promote mrnas entering P-odies 5 7. Thus, the presence of mirnatrgeted mrnas in P-odies rgues tht if RISC inhiits trnsltion directly, it is likely to do so t the level of trnsltion initition. An lterntive model is tht locliztion to the P-ody is direct consequence of the interction etween the Argonute protein/mirna complex nd its trget, perhps y Argonute promoting the ssemly of trnsltionlly repressed mrnp trgeted to P-ody. In this model, it is the locliztion to the P-ody tht is, itself, responsile for preventing protein synthesis, ecuse the trnsltion mchinery is excluded from this structure in oth yest nd mmmls 6,29. A hyrid model suggests tht RISC-medited trnsltion repression involves n initil inhiition of trnsltion per se, which then leds to trgeting of the mrna to P-ody, where sequestrtion from the trnsltion mchinery reinforces silencing. Note dded in proof: After this study hd een ccepted for puliction, Sen nd Blu 30 reported similr oservtions of the locliztion of humn to cytoplsmic P-odies. METHODS DNA constructs. -tgged Ago expression plsmids were s descried in ref. 16. GFP-tgged, Flg-tgged nd plsmids were s descried in ref. 4. -PAZ9 nd -PAZ10 mutnts contined multiple point muttions in the PAZ domin of (R277A, K278A, Y279A, F294A, Y311A, F312G, T337A, Y338A, L339A, /+ H271A). Muttions were introduced y site-directed mutgenesis using the QuickChnge Kit from Strtgene (L Joll, CA). The mrna reporters were constructed y modifying the pcdna3-firefly luciferse construct s descried in Fig. 4. The let-7 mirna recognition sites were derived from C. elegns lin-41 3 UTR. The CXCR4 sirna recognition sites were derived from CXCR4 sirna-response luciferse reporter s descried in ref. 22. Cell culture nd trnsfection. Humn U2-OS, HeL nd 293T cells were cultured in DMEM (10% FBS) in 37 C incutor with 5% CO 2. Cell trnsfections were performed using Mirus (Mdison, WI) TrnsIT-LT1 regent for DNA plsmids nd Invitrogen (Crlsd, CA) Oligofectmine regent for sirnas. Procedures for immunoprecipittion, immunolotting, RNA extrction, smll RNA lotting nd slicer ssy were descried previously 16. To ssess the RNA dependence of protein protein interctions, lystes were treted with RNAseA t 0.5 µg µl 1 for 20 min t room temperture efore immunoprecipittion procedures s descried in ref. 18. Immunofluorescence. Cultured cells were fixed using 3% prformldehyde (in PBS) for 12 min t room temperture. The cells were then permeilized in PBS contining 0.2% Triton-X100 for 6 min t 4 C. FITC- or Rhodmine-Red-conjugted secondry ntiodies were purchsed from Jckson Immunoreserch (West Grove, PA). Antiody incutions were done t room temperture in PBS contining 0.5% BSA s locking gent. The cells were exmined using n Axioskop (Crl Zeiss, Thornwood, NY) fluorescent microscope. In trnsfected cells, not ll cells showed foci; for exmple, in experiments following locliztion of mirna trgets, roughly 20% of cells showed tht mirna trgets re in foci. This is proly ecuse of the need to chieve pproprite expression levels of trget RNA nd two exogenous proteins. BIND identifiers. Four BIND identifiers ( re ssocited with this mnuscript: , , nd Note: Supplementry Informtion is ville on the Nture Cell Biology wesite. ACKNOWLEDGEMENTS We thnk memers of the Hnnon lortory for helpful discussions, S. Hern from the CSHL microscopy shred resource for ssistnce, nd S. Jnicki (CSHL), J. Lykke-Andersen (University of Colordo) nd T. Achsel (University of Wurzurg) for regents. J.L. is supported y Specil Fellow wrd from the Leukemi nd Lymphom Society. This work ws supported y grnts from the NIH (to G.J.H. nd R.P.). R.P. is n investigtor t the Howrd Hughes Medicl Institute. COMPETING FINANCIAL INTERESTS The uthors declre tht they hve no competing finncil interests. Received 26 April 2005; ccepted 20 My 2005 Pulished online t 1. Hnnon, G. J. RNA interference. Nture 418, (2002). 2. Ingelfinger, D., Arndt-Jovin, D. J., Luhrmnn, R. & Achsel, T. The humn LSm1-7 proteins coloclize with the mrna-degrding enzymes Dcp1/2 nd Xrnl in distinct cytoplsmic foci. RNA 8, (2002). 3. vn Dijk, E. et l. Humn : ctlyticlly ctive mrna decpping enzyme locted in specific cytoplsmic structures. EMBO J. 21, (2002). 4. Lykke-Andersen, J. Identifiction of humn decpping complex ssocited with hupf proteins in nonsense-medited decy. Mol. Cell. Biol. 22, (2002). 5. Sheth, U. & Prker, R. Decpping nd decy of messenger RNA occur in cytoplsmic processing odies. Science 300, (2003). 6. Teixeir, D., Sheth, U., Vlenci-Snchez, M. A., Brengues, M. & Prker, R. Processing odies require RNA for ssemly nd contin nontrnslting mrnas. RNA 11, (2005). 7. Cougot, N., Bjko, S. & Serphin, B. Cytoplsmic foci re sites of mrna decy in humn cells. J. Cell. Biol. 165, (2004). 8. Meister, G. & Tuschl, T. Mechnisms of gene silencing y doule-strnded RNA. Nture 431, (2004). 9. Wightmn, B., H, I. & Ruvkun, G. Posttrnscriptionl regultion of the heterochronic gene lin-14 y lin-4 medites temporl pttern formtion in C. elegns. Cell 75, (1993). 10. Lee, R. C., Feinum, R. L. & Amros, V. The C. elegns heterochronic gene lin-4 encodes smll RNAs with ntisense complementrity to lin-14. Cell 75, (1993). 11. Ketting, R. F. et l. Dicer functions in RNA interference nd in synthesis of smll RNA involved in developmentl timing in C. elegns. Genes Dev. 15, (2001). 12. Knight, S. W. & Bss, B. L. A role for the RNse III enzyme DCR-1 in RNA interference nd germ line development in Cenorhditis elegns. Science 293, (2001). 13. Hutvgner, G. et l. A cellulr function for the RNA-interference enzyme Dicer in the mturtion of the let-7 smll temporl RNA. Science 293, (2001). 14. Brtel, D. P. MicroRNAs: genomics, iogenesis, mechnism, nd function. Cell 116, (2004). 15. Amros, V. The functions of niml micrornas. Nture 431, (2004). 16. Liu, J. et l. Argonute2 is the ctlytic engine of mmmlin RNAi. Science 305, (2004). 17. Eystthioy, T. et l. The GW182 protein coloclizes with mrna degrdtion ssocited proteins hdcp1 nd hlsm4 in cytoplsmic GW odies. RNA 9, (2003). 18. Thrun, S. et l. Yest Sm-like proteins function in mrna decpping nd decy. Nture 404, (2000). 19. M, J. B., Ye, K. & Ptel, D. J. Structurl sis for overhng-specific smll interfering RNA recognition y the PAZ domin. Nture 429, (2004). 20. Jnicki, S. M. et l. From silencing to gene expression: rel-time nlysis in single cells. Cell 116, (2004). 21. Lewis, B. P., Shih, I. H., Jones-Rhodes, M. W., Brtel, D. P. & Burge, C. B. Prediction of mmmlin microrna trgets. Cell 115, (2003). 22. Doench, J. G., Petersen, C. P. & Shrp, P. A. sirnas cn function s mirnas. Genes Dev. 17, (2003). 23. Souret, F. F., Kstenmyer, J. P. & Green, P. J. AtXRN4 degrdes mrna in Aridopsis nd its sustrtes include selected mirna trgets. Mol. Cell 15, (2004). 24. Orn, T. I. & Izurrlde, E. Decy of mrnas trgeted y RISC requires XRN1, the Ski complex, nd the exosome. RNA 11, (2005). 25. Newury, S. & Woollrd, A. The 5-3 exorionuclese xrn-1 is essentil for ventrl epithelil enclosure during C. elegns emryogenesis. RNA 10, (2004). 26. Gzzni, S., Lwrenson, T., Woodwrd, C., Hedon, D. & Slowski, R. A link etween mrna turnover nd RNA interference in Aridopsis. Science 306, (2004). 27. Lim, L. P. et l. Microrry nlysis shows tht some micrornas downregulte lrge numers of trget mrnas. Nture 433, (2005). 28. Jing, Q. et l. Involvement of microrna in AU-rich element-medited mrna instility. Cell 120, (2005). 29. Andrei, M. A. et l. A role for eif4e nd eif4e-trnsporter in trgeting mrnps to mmmlin processing odies. RNA 11, (2005). 30. Sen, G. L. & Blu, H. M. Argonute 2/RISC resides in sites of mmmlin mrna decy known s cytoplsmic odies. Nture Cell Biol. 7, (2205). NATURE CELL BIOLOGY VOLUME 7 NUMBER 7 JULY

6 SUPPLEMENTARY INFORMATION myc-(h634a) myc-(d597a) myc-(d669a) Figure S1 Locliztion of ctlyticlly inctive mutnts. Ctlyticlly inctive mutnts, H634A, D597A nd D669A were expressed in U2-OS cells s myc-tgged proteins nd loclized in cells using FITC-conjugted nti-myc 1

7 SUPPLEMENTARY INFORMATION myc- myc-ago3 myc-ago4 Figure S2 Locliztion of ctlyticlly inctive Argonute sufmily proteins. -tgged, Ago3 nd Ago4 proteins were immunoloclized in humn U2- OS cells using FITC-conjugted nti-myc ntiodies. 2

8 SUPPLEMENTARY INFORMATION mrna reporter: MS2-YFP-NLS myc-ago3 merge YFP MS2 MS2 YFP let7 mirna YFP MS2 MS2 YFP MS2-YFP-NLS myc-ago3 merge exogenous CXCR4 sirna Figure S3 Co-locliztion of Ago3 with mirna trgets. () The luciferse-lin41 UTR fusion reporter ws co-expressed in U2-OS cells with n MS2-YFP-NLS fusion protein nd myc-ago3. Reporter mrna ws visulized indirectly with YFP. Ago3 ws loclized using Rhodmine Red conjugted nti-myc ntiody. () The luciferse-cxcr4 reporter ws expressed in cells tht were lso trnsfected with the CXCR4 sirna nd plsmid directing the expression of myc- Ago3. Anlysis ws s in. 3