A conserved PUF Ago eef1a complex attenuates translation elongation

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1 A onserved PUFAgoeEFA omplex ttenutes trnsltion elongtion Kyle Friend, Zhry T Cmpell, Amy Cooke,, Peggy Kroll-Conner, Mrvin P Wikens, & Judith Kimle Nture Ameri, In. All rights reserved. PUF (Pumilio/FBF) RNA-inding proteins nd Argonute (Ago) mirna-inding proteins regulte mrnas post-trnsriptionlly, eh ting through similr, yet distint, mehnisms. Here, we report tht PUF nd Ago proteins n lso funtion together in omplex with ore trnsltion elongtion ftor, eefa, to repress trnsltion elongtion. Both nemtode (Cenorhditis elegns) nd mmmlin PUFAgoeEFA omplexes were identified, using oimmunopreipittion nd reominnt protein ssys. Nemtode CSR- (Ago) promoted repression of FBF (PUF) trget mrnas in in vivo ssys, nd the FBF-CSR- heterodimer inhiited EFT- (eefa) GTPse tivity in vitro. Mmmlin AgoeEFA inhiited trnsltion of nondenylted nd polydenylted reporter mrnas in vitro. This repression ourred fter trnsltion initition nd led to riosome umultion within the open reding frme, roughly t the site where the nsent polypeptide emerged from the riosoml exit tunnel. Together, these dt suggest tht onserved PUFAgoeEFA omplex ttenutes trnsltion elongtion. Trnsltionl regultion ffets metzon development, lerning nd humn disese. Of the mny RNA-inding proteins regulting mrna stility, loliztion nd trnsltion, the two lsses relevnt to this work re the PUF nd Ago fmilies. PUF proteins regulte rod spetrum of mrnas to mintin stem ells, mong other funtions,. Ago proteins ind smll RNAs, most notly mirnas, nd t in mny iologil ontexts, inluding in stem ells (reviewed in ref. ). PUF proteins regulte mrna expression in virtully ll eukryotes. One onserved mehnism, whih hs een estlished for Shromyes erevisie, Cenorhditis elegns nd humn PUF proteins, relies on the diret reruitment of the CrNot dedenylse omplex to trget mrnas, resulting in shorter poly(a) til length nd either mrna instility or trnsltionl repression,6. PUF proteins in yest nd flies lso use dedenyltion-independent mehnisms, lthough the speifis of these mehnisms remin mostly unknown 7,8. Ago proteins funtion within the mirisc omplex to ontrol trget mrna stility nd trnsltion. This omplex ontrols dedenyltion nd stility of its trget mrnas 9, ut its mehnism to ontrol trnsltion hs grnered ontroversy. Best doumented is its regultion of trnsltion initition 9. However, mehnism ffeting trnsltion elongtion lso ppers to exist, s trget mrnas n e ound to polyriosomes. One model is tht mirisc promotes riosoml drop-off during trnsltion elongtion. Yet the speifi mehnism y whih mirisc, nd hene Ago fmily memers, inhiits trnsltion elongtion is not understood. Here, we report tht PUF nd Ago proteins form n inhiitory omplex with eefa, GTPse required for trnsltion elongtion. FBF- ( C. elegns PUF protein) inds CSR- ( C. elegns Ago fmily memer) in vitro nd in vivo, nd sr- depletion leds to inresed expression of FBF trget mrnas. The FBF-CSR- heterodimer forms omplex with EFT- (C. elegns eefa), nd this FBF-CSR- EFT- ternry omplex hs inhiited GTPse tivity. Notly, the PUFAgoeEFA omplex is onserved: humn ( PUF protein) ssoites with humn AGO proteins in vivo nd with eefa. Wild-type humn inhiits trnsltion of oth nondenylted nd polydenylted mrnas in rit retiuloyte lyste; however, mutnts tht nnot form the AgoeEFA omplex or tht nnot ind RNA re severely ompromised for trnsltion repression. Mehnistilly, AgoeEFA represses trnsltion during elongtion, with riosomes umulting ~ nuleotides (nt) fter the AUG within the open reding frme (ORF). We propose model in whih PUF nd AGO proteins form omplex with eefa to inhiit its GTPse tivity nd ttenute trnsltion elongtion. RESULTS Assoition of C. elegns FBF- nd CSR- To explore the moleulr mehnisms of PUF protein funtion, we sought proteins tht intert with two nerly identil C. elegns PUF proteins, FBF- nd FBF-, known olletively s FBF 6. Previous yest two-hyrid sreens identified severl FBF prtners ut no omponents of the trnsltion mhinery 7,8. Here, we used reominnt, fulllength GSTFBF- nd GSTFBF- to selet proteins from C. elegns lystes nd to identify interting proteins y mss spetrometry (Supplementry Fig. nd Supplementry Tle ). Known FBFinterting proteins, CPB- nd NOS- (refs. 7,8), were deteted in Deprtment of Biohemistry, University of Wisonsin-Mdison, Mdison, Wisonsin, USA. Grdute Progrm in Cellulr nd Moleulr Biology, University of Wisonsin-Mdison, Mdison, Wisonsin, USA. Howrd Hughes Medil Institute, University of Wisonsin-Mdison, Mdison, Wisonsin, USA. Correspondene should e ddressed to J.K. (jekimle@wis.edu). Reeived 8 July ; epted Novemer ; pulished online 8 Jnury ; doi:.8/nsm. nture struturl & moleulr iology dvne online pulition

2 Nture Ameri, In. All rights reserved. Figure FBF- inds CSR- to repress trget mrna. () FBF- oimmunopreipittes with CSR-. Trnsgeni GFPøFBF- (FBF-) ws immunopreipitted from dult hermphrodites with or without RNse. Bound proteins (pellets) were proed on western lots for indited proteins. CSR- nd EFT- were oth deteted in FBF- immunopreipittions, wheres DRH- nd tin were not. Two CSR- isoforms exist in vivo,, nd oth oimmunopreipitte with FBF-. CSR- did not oimmunopreipitte with GFPøtuulin (Tu). () CSR- is expressed in the mitoti zone. Top, digrm of sr- lous. The q79 deletion removes ruil domins, resulting in frmeshift. Arrows, lterntive promoter elements. Bottom: CSR- ntiody stins the wild-type mitoti zone ytoplsm (, distl end of the gond; dshed line, mitoti zonetrnsition zone oundry), ut not in sr- mutnt. (,d) Adult germ lines expressing GFPøHB (green) under ontrol of wildtype (WT) or FBE-lking ( FBE) gld- UTR; nulei seen with DAPI (lue). Mrked inlude:, distl end; open tringle, distl-most GFPpositive ell; losed tringle, distl-most right GFP; dshed line, mitoti zonetrnsition zone oundry. Control RNAi, empty vetor. GFPøHB is normlly repressed in distl region (top pnels), ut expnds into distl germ ells without FBEs (lower pnels) (). sr-(rnai). GFPøHB expnded more distlly thn ontrol in pnel when reporter hrors n FBE (d). (e) GFPøHB extent fter RNAi ginst genes indited, soring only sterile nimls to ensure effetive RNAi. Blue olumns, most distl germ-ell row with GFPøHB-positive ell; red olumns, mitoti zone trnsition zone oundry. Error rs represent s.d. (n > ). (f) Perent ells in mitoti zone with right GFPøHB. ddition to previously unknown puttive prtners CSR-, one of 7 C. elegns Ago fmily memers 9,, nd EFT-, the C. elegns homolog of trnsltion elongtion ftor A, eefa. To diretly test whether FBF- interts with CSR- nd EFT- in vivo, we immunopreipitted tgged FBF- (ref. ) from C. elegns lyste nd proed for ssoited proteins. CSR- ws speifilly enrihed, nd this intertion ws RNA-independent (Fig. ), inditing tht FBF- nd CSR- form protein omplex. By ontrst, Dier-relted helise (DRH-), whih is required to produe the -nt RNAs ound y CSR- (refs.,), did not oimmunopreipitte with FBF- (Fig. ), demonstrting tht FBF- speifilly inds CSR-. EFT- ws present in oth FBF- nd ontrol oimmunopreipittions (Fig. ), so we rried out further ssys to onfirm speifiity of inding (see elow). Our oimmunopreipittion experiments suggested tht CSR- should e expressed in the sme germline region s FBF. FBF is enrihed in the germ-ell ytoplsm t the distl end of the gond, nd we found CSR- in the sme lotion (Fig. ), in ddition to its known lotion in the proximl germ line. Beuse FBF- nd CSR- oimmunopreipitte nd re expressed in the sme region, FBF- nd CSR- proly intert in vivo. CSR- promotes repression of FBF trget mrnas PUF nd Ago fmily memers oth repress trnsltion,, nd models hve suggested tht mirna inding sites proximl to PUF mrna trget sites my indite n intertion 6. To sk if FBF nd CSR- might funtion together, we exmined expression of n FBF reporter mrna fter CSR- depletion. In the reporter mrna, the UTR of gld-, n FBF trget mrna, ws linked to n ORF enoding GFP histone HB fusion protein (GFPøHB), whih lels nulei. We used RNAi to deplete sr- from nimls trnsgeni for reporters with either the wild-type gld- UTR (WT gld- UTR) or mutnt gld- UTR ( FBE gld- UTR) without FBF inding elements (FBEs) 7. Briefly, fourth-stge lrve were treted y feeding RNAi, nd the GFPøHB reporter ws sored one genertion lter in dult progeny. In ontrol nimls not depleted for CSR-, GFPøHB ws sent from the distl-most germ ells with the WT gld- UTR reporter (Fig., top pnels), ut GFPøHB extended to the distl GFP:: GFP CSR- DRH- EFT- WT gld- UTR FBE gld- UTR Atin % Input Tu FBF- Control RNAi Pellets Tu FBF- RNse d sr- sr- (RNAi) end in nimls with the FBE gld- UTR reporter (Fig., ottom pnels), s seen previously 7. After CSR- depletion in nimls rrying the WT gld- UTR reporter, right GFPøHB expression ws lwys shifted distlly ompred to ontrols (n = /) (Fig. d, top pnels) nd ws often in distl-most germ ells (n = /). By ontrst, sr- RNAi hd little effet on expression of the FBE gld- UTR reporter (Fig. d, ottom pnels). As ontrol, the sme reporter ssy ws used fter proteins thought to form omplex with CSR- (ref. ) were depleted, ut these depletions did not ffet the WT gld- UTR reporter (Fig. e,f). Beuse totl loss of FBE-medited repression lwys leds to right GFPøHB in the distl-most germ ells 8, sr- depletion does not ompletely eliminte FBE-medited repression ut insted severely ompromises tht repression. We onlude tht CSR- redues expression of FBF reporter mrna nd tht this effet requires FBEs. To vlidte our findings with the gld- reporter mrnas, we ssyed two FBF trget mrnas, gld- (ref. ) nd ye- (ref. 9) mrnas, in sr- mutnt. GLD- protein is normlly expressed t low ut grded level in the mitoti zone (Supplementry Fig., top pnel), wheres CYE- is more uniform in the mitoti zone (Supplementry Fig., middle pnel). In sr- deletion mutnt, GLD- nd CSR- oth inresed mrkedly in the mitoti zone, though GLD- ws still grded (Supplementry Fig., top nd middle pnels). For ontrols, we lso nlyzed GLD- nd CYE- levels in mutnts of drh-, ego- nd ekl-, whih re thought to funtion jointly with sr- (refs.,,). In these mutnts, we oserved more GLD- nd CYE- thn wild-type, ut not to the high level typil of sr- mutnts (Supplementry Fig. e). Therefore, wild-type CSR- lowers expression of t lest two FBF trget mrnas. These experiments suggest tht FBF funtions with CSR- to repress trget mrnas. Yet, the doumented germline defets in ff nd sr- mutnts differ in mny respets. The ff- ff- doule mutnt germ lines re defetive for germline self-renewl nd hene hve no mitoti zone wheres sr- is defetive for hromosome segregtion nd emryoni viility 9. We exmined sr- deletion mutnts for mitoti zone defets nd found the sr- mitoti zone to e present ut smller thn norml (Supplementry Fig. ). The redued sr- mitoti Wild-type sr-(q79) PAZ MID PIWI e fgerm ell dimeters from distl end Nulei in MZ with right GFP (%) q79 deletion nti-csr- 8 6 Distl-most right GFP MZ/TZ oundry Control nti-csr- Control sr- drh- ego- ekl- sr- drh- ego- ekl- dvne online pulition nture struturl & moleulr iology

3 GSTFBF- His 6 -MID-PIWI Glutthione Ni GSTFBF- His 6 -PAZ Glutthione Ni FBF- FBF-(FR) CSR- EFT- FBF- CSR- EFT- Inputs Pellets d. P i hydrolyzed per EFT- (fmol).... EFT- EFT- (H9L) EFT- FBF- EFT- FBF- CSR- EFT- FBF-(FR) CSR- Figure FBF-CSR-EFT- ternry omplex formtion redues EFT- GTPse tivity. () GSTFBF- nd His 6 -CSR-(MID-PIWI) were oexpressed in the petduet system. Affinity seletion ws rried out with either glutthione to selet GSTFBF- or Ni to selet His 6 -MID-PIWI, respetively. GSTFBF- nd His 6 -MID-PIWI ssoite. His 6 -MID-PIWI degrdtion produts re indited (). () As in pnel, ut GSTFBF- ws oexpressed with CSR- PAZ (His 6 -PAZ). FBF- nd His 6 -PAZ do not ssoite. () EFT- inds the FBF-CSR- omplex. Reominnt EFT- ws dded to wildtype FBF-, FBF-(FR), FBF-CSR- or FBF-(FR)CSR- prepred from teri (inputs, % loded). Glutthione ws used to selet GST FBF- (wild-type nd mutnt) fter inution (pellets, % loded). EFT- (rrowhed) speifilly ssoited with wild-type FBF-CSR-, ut not with FBF- lone or with mutnt FBF-(FR)CSR-. We note tht the FBF-CSR-EFT- omplex forms with pprent :: stoihiometry. CSR- refers to His 6 -CSR-(MID-PIWI); FBF- refers to GSTFBF-. (d) FBF-CSR- speifilly inhiits EFT- GTPse tivity. [γ- P]GTP ws inuted with EFT-, tlytilly ded EFT-(H9L), EFT- nd FBF-, EFT-FBF-CSR- or EFT- nd FBF-(FR)CSR-. Lierted P i ws ounted fter phse extrtion. Error rs represent s.d. (n = ). Nture Ameri, In. All rights reserved. zone is onsistent with funtionl overlp with FBF, ut its presene demonstrtes tht CSR- is not required for ll FBF funtions. We suggest tht CSR- nd FBF n work together to repress mrna expression through FBEs, ut tht they n lso work independently. The independent funtions proly involve intertions with other prlogs (for exmple, FBF with other Agos nd CSR- with other PUFs) s well s intertions with other omplexes (for exmple, FBF with CrNot dedenylse nd CSR- with RISC). FBF- interts with the CSR- MID-PIWI domins To diretly ssy FBF- intertions with CSR-, we onduted iohemil nlyses with purified reominnt proteins. Initilly, we explored the intertion, fousing on onserved domins the PUF repets of FBF- (herefter lled FBF-) nd the MID-PIWI or PAZ domins of CSR-. GSTFBF- (residues 6) ws oexpressed with either His 6 -MID-PIWI (residues 799) or His 6 -PAZ (residues 78) in the petduet system, whih llows simultneous expression of multiple proteins. Affinity seletion for GSTFBF- enrihed His 6 -MID-PIWI (Fig., lne ) ut not His 6 -PAZ (Fig., lne ); the reiprol seletion for His 6 -MID-PIWI enrihed GSTFBF- (Fig., lne ), ut seletion for His 6 -PAZ did not enrih GSTFBF- (Fig., lne ). Therefore, purified FBF- interts speifilly with the CSR- MID-PIWI domins ut not with its PAZ domin. Our next experiments used the CSR- MID-PIWI region, referred to s CSR- for revity. FBF- nd CSR- form omplex with EFT- (eefa) This work identifies EFT- s potentil FBF- intertor (Supplementry Tle ), nd work y others identified humn eefa s potentil AGO nd AGO intertor, lthough the intertion ws not onfirmed for either nemtode or humn omponents. We hypothesized tht the FBF-CSR- heterodimer might form ternry omplex with the trnsltion elongtion ftor EFT-. To explore tht ide, we purified FBF- lone, the FBF-CSR- heterodimer nd EFT- lone (Fig., lnes ); ttempts to purify CSR- y itself were unsuessful. FBF- lone did not ind EFT- (Fig., lne ), ut the FBF-CSR- heterodimer did ind EFT- (Fig., lne 7), suggesting the existene of ternry omplex. This ternry omplex ws oserved in the sene of GTP (Fig., lne 7), ut FBF-CSR- lso ound EFT- when GTP ws inluded (Supplementry Fig. ). We onfirmed the identity of CSR- nd EFT- proteins in these pull-down ssys y western lotting (Supplementry Fig. ). We exmined FBF- for onserved residues tht re not involved in RNA-inding nd found rodly onserved phenyllnine residue (Phe) (Supplementry Fig. ). We hypothesized tht mutnt FBF-(FR) might e defetive for ternry omplex formtion. Although FBF-(FR) ws le to ind CSR- (Supplementry Fig. d), the FBF-(FR)CSR- heterodimer filed to ind EFT- (Fig., lne 8). We propose tht FBF-CSR-EFT- exists s omplex. The FBF-CSR- heterodimer inhiits EFT- GTPse tivity How might the omplex ffet trnsltion? eefa fmily memers possess GTPse tivity, whih is required during trnsltion elongtion in order to relese minoyl-trnas upon delivery to the riosome. Purified eefa hs intrinsi GTPse tivity, whih is stimulted y riosomes. We hypothesized tht the PUFAgo omplex might inhiit eefa tivity euse PUF nd Ago proteins n inhiit trnsltion,9. To test this ide, we first onfirmed tht wild-type EFT- possesses GTPse tivity ompred to tlytilly ded mutnt EFT-(H9L) (Fig. d; Supplementry Fig. e). We then ssyed FBF- lone nd the FBF-CSR- heterodimer for n effet on the EFT- GTPse tivity. FBF- lone did not redue GTPse tivity, ut the FBF-CSR- heterodimer redued GTPse tivity to level similr to tht of tlytilly ded EFT-(H9L) (Fig. d). Notly, the mutnt FBF-(FR)CSR- heterodimer, whih did not form omplex with EFT-, lso did not inhiit EFT- GTPse tivity (Fig. d). Inhiition of EFT- GTPse tivity in the FBF-CSR-EFT- omplex suggests tht the omplex my repress trnsltionl elongtion. A humn AGOeEFA omplex We predited tht AGOeEFA omplex might lso form in humns. Initilly, we tested whether inds AGO fmily memers in vivo. HEK9T ells were trnsiently trnsfeted with onstruts enoding the Flg-tgged PUF repets of humn (- Flg) nd Flg-hemgglutinin (HA)-tgged, full-length humn AGOs (ref. ). Using nti-ha ntiodies, AGOs were immunopreipitted from ells ross-linked with formldehyde under denturing onditions with RNse to isolte endogenous omplexes. Anti-Flg western lots reveled tht nd AGOs oimmunopreipitte (Supplementry Fig. ). We onfirmed tht oimmunopreipittes with AGOs without ross-linking (Supplementry Fig. ). To test whether is ritil for the nture struturl & moleulr iology dvne online pulition

4 Nture Ameri, In. All rights reserved. Figure AgoeEFA omplex inhiits protein prodution. () Humn promotes eefa ssoition with Ago. Western lots using Flg ntiody to detet Flg- nd Flg-HA-AGOs (top) or eefa ntiody (ottom). oexpression mrkedly enhned eefa oimmunopreipittion. () RNA inding y vrints. Reominnt, (F868R), (T7E) nd (H8A) t inresing onentrtions (tringle) were ssyed for inding the hunhk PBE sequene. () Complex formtion with vrints. AGO opreipittes with ll mutnts exept (T7E). eefa opreipittes with wild-type nd (H8A) ut not with (F868R) or (T7E). Equivlent mounts of reominnt proteins were dded to retiuloyte lyste (input) in the presene of RNse. (d) vrint summry. (e) PBE-dependent trnsltion repression of nondenylted reporter mrna. Reominnt GST or GST-tgged vrints, nondenylted firefly luiferse mrna with PBEs in its UTR nd ontrol nondenylted Renill luiferse reporter were dded to retiuloyte lyste. To monitor PBE-dependent trnsltion, firefly luiferse prodution ws normlized to Renill luiferse prodution. Only wild-type inhiited trnsltion of nondenylted firefly luiferse mrna. Note tht protein output ws not orreted for mrna level, euse ll retion mixtures ontined the sme initil quntity of reporter mrnas, nd wildtype retion mixtures hd more finl mrna thn others. Error rs represent s.d. (n = ). (f) As in pnel e, ut with polydenylted mrnas. ssoition of Ago nd eefa, we onduted the sme trnsfetion experiments s ove in the presene or sene of. When ws oexpressed, AGO oimmunopreipittions pulled down mrkedly more eefa thn when ws omitted, suggesting tht AGO inds eefa in vivo (Fig. ). We onlude tht the AGOeEFA omplex is onserved. mutnts olish omplex formtion We turned to rit retiuloyte lyste to hrterize AgoeEFA effets on trnsltion. For these experiments, we first identified mutnts tht olish omplex formtion or RNA inding. Eh vrint tested ws omposed of the PUF repets of humn (residues 766) nd ws generted s GST fusion protein. refers to the wild-type version; (F868R) lters the onserved phenylnine orresponding to FBF-(F); (T7E) hnges onserved threonine residue (Supplementry Fig. ); nd (H8A) ws predited to rogte RNA inding. Of these four proteins (Supplementry Fig. d), only (H8A) filed to ind n RNA rrying PUF-inding elements (PBEs) (Fig. ). When ssyed for omplex formtion, wild-type nd (H8A) opreipitted Ago nd eefa from rit retiuloyte lyste (Fig., lnes nd 6). (F868R) ound Ago, ut not eefa (Fig., lne ), nd (T7E) filed to ind either Ago or eefa (Fig., lne ). In summry, (T7E) is Ago-inding defetive (ABD); (F868R) is eefa-inding defetive (EBD); nd (H8A) is RNA-inding defetive (RBD) ut inds oth Ago nd eefa (Fig. d). The Ago heterodimer reruits eefa, nd RNA-inding tivity is not required for omplex formtion. AgoeEFA represses mrna trnsltion To ssy effets of the AgoeEFA omplex on trnsltion, we used reporter mrnas in retiuloyte lyste. We first quntitted Ago levels in the lyste (Supplementry Fig. 6) nd dded n equivlent mount of. To ensure tht retiuloyte lyste ws not limiting for trnsltion, we titrted mrna enoding firefly luiferse with PBEs in its UTR (PBEs ind humn PUM nd (refs. 6,7)) ginst onstnt ontrol Renill luiferse reporter lking PBEs (Supplementry Fig. 6). did not destilize reporter Bound RNA d e PBE-dependent trnsltion (firefly or Renill tivity) Free RNA vrint Nondenylted mrnas 8 6 AGO Flg-HA-AGO -xflg eefa (T7E) (F868R) (H8A) GST (F868R) (T7E) (H8A) Ago inding ABD Inputs RNA inding EBD RBD mrnas; in ft, stilized them (Supplementry Fig. 6). Therefore, the rtio of firefly to Renill luiferse enzyme tivities llows us to monitor PBE-dependent trnsltion. We first ssyed nondenylted mrna (Fig. e) to exlude from our nlysis PUF-medited dedenyltion effets. Wild-type inhiited mrna trnsltion, ut the three mutnt proteins filed to repress reporter expression (Fig. e). Most notly, the ABD nd EBD mutnts, whih disrupt PUFAgoeEFA omplex formtion (Fig. ), did not repress the reporter. Most nturl mrnas re polydenylted, so we lso ssyed polydenylted reporter mrnas (Fig. f). Similr to the effets oserved on the nondenylted reporter mrna, wild-type repressed trnsltion of polydenylted mrna wheres (RBD) did not. Notly, the ABD nd EBD mutnts remined ple of limited repression. One possiility might hve een tht the ABD nd EBD mutnts hd this minor effet y promoting mrna dedenyltion. However, mrna denyltion ws unffeted in our ssys (Supplementry Fig. 6d). An open question is how (ABD nd EBD) mutnts repress the polydenylted reporter in the sene of oth dedenyltion nd AgoeEFA omplex formtion. Indeed, these mutnts suggest the existene of yet nother mehnism of PUF repression. Beuse PUF proteins ssoite with the protein nnos 8, we onsidered the possiility tht the region of importnt for its nnos ssoition might e importnt for repression. However, (G987D) repressed oth nondenylted nd polydenylted reporter mrnas s well s wild-type (Supplementry Fig. 7). Together, results from experiments using nondenylted nd polydenylted mrna reporters suggest tht the PUFAgoeEFA omplex represses trnsltion nd tht it does so independent of mrna dedenyltion. f AGO eefa eefa inding PBE-dependent trnsltion (firefly or Renill tivity) 6 GST Input Wild-type GST Summry ABD EBD (F868R) (T7E) (H8A) Ago-inding defetive (ABD) eefa-inding defetive (EBD) RNA-inding defetive (RBD) Adenylted mrnas nti-ha pellets RBD dvne online pulition nture struturl & moleulr iology

5 Nture Ameri, In. All rights reserved. Figure Kinetis of trnsltionl repression. (,) proteins were inuted with reporter mrnas in retiuloyte lyste for min to ensure riosome loding; t time t =, exess mrna p nlog ws dded to inhiit de novo S riosoml suunit loding. Luiferse prodution ws monitored every seonds. Error rs represent s.d. (n = ). () Renill luiferse prodution. Left, kinetis of Renill luiferse prodution were equivlent for nd (RBD); right, enlrgement of erly time points. () Firefly luiferse prodution. Left, firefly luiferse prodution ws delyed with ompred to (RBD) nd mrkedly inhiited midwy through the retion. The urve ws similr to tht of (RBD) from the ~- to 8-min time points (leit with -s dely), suggesting tht fully funtionl repressive omplex forms slowly. Right, enlrgement of dt points when firefly luiferse is first produed. () To estimte trnsltionl elongtion rte, we ompred the time required to first produe either firefly or Renill luiferse (firefly luiferse is 9 residues longer thn Renill luiferse). In retion mixtures with (RBD), the first firefly luiferse protein ws deteted. min fter the first Renill luiferse (. min for firefly versus min for Renill). From this differene, we infer n estimted trnsltionl elongtion rte (eter) of. residues per seond (9/ residues s ). In retion mixtures with wild-type, the sme lultion yields n eter of.77 residues per seond. This modest derese ws seen t time well efore eme fully repressive (8 min fter p nlog ddition). Therefore, my ffet trnsltion elongtion y using multiple mehnisms. AgoeEFA represses trnsltion fter initition We next investigted the mehnism of trnsltion inhiition, gin in rit retiuloyte lystes. Initilly, we rried out stndrd ssy for inhiition of trnsltion initition: we frtionted polyriosomes to sk if inhiits the ssoition of its trget mrna with riosomes. In vitro trnsltion retion experiments were onduted s ove with firefly mrna ( PBEs) nd Renill mrna (ontrol) in the presene of or (RBD). Retion mixtures were quenhed with yloheximide (mintins riosomes on mrna) or puromyin (removes riosomes from mrna) nd seprted over surose grdient. After entrifugtion, polyriosome profiles were indistinguishle when or (RBD) ws dded, lthough puromyin did disrupt polyriosomes (Supplementry Fig. 8,). Moreover, y northern lots, the PBE-ontining mrna ssoited with polyriosomes to the sme level s the ontrol mrna, if either or (RBD) ws dded (Supplementry Fig. 8). As the PBE-ontining mrna migrted in lighter frtions fter puromyin ddition, it is ound to riosomes (Supplementry Fig. 8d). Although these dt ontrst oservtions tht mirna-medited trnsltion repression ours t initition in rit retiuloyte lyste 9, we note tht we investigted PUFAgoeEFA, not mirnas. Therefore, riosoml ssoition with -ound mrna ppers unffeted y this ssy, leding us to sk further questions out how trnsltion is inhiited. We next vried the trnsltion ssys to sk if -medited repression ours fter trnsltion initition. Reporter RNAs plus purified reominnt protein were inuted in lyste for min to egin trnsltion; then exess mrna p nlog ws dded to quenh dditionl riosoml loding nd thus to lok trnsltion initition. Prodution of firefly (whih is sujet to regultion) nd Renill luiferse ws monitored in the presene of either or (RBD) over time. In the sene of the p nlog, reporters produed luiferse linerly for out min; however, in the presene of the p nlog, luiferse prodution stopped muh erlier. The ontrol Renill luiferse RNA plteued fter only ~ min in the presene of either or (RBD) (Fig. ). This erly plteu indited tht the exess p nlog hd olished de novo S riosoml suunit loding. With the RNA-inding defetive Renill luiferse units ( ) Firefly luiferse units ( 6 ) (RBD) Time (min) (RBD) Time (min) nd RNA dded to lyste Aumultion of Renill first seen (± ) Cp nlog quenhes riosome loding Riosome loding Aumultion of firefly first seen (RBD) (WT) 6 Time (min) 8 7 (RBD) Time (min) vrint, expression from the firefly luiferse mrna plteued fter ~ min, it fter the Renill ontrol, presumly euse the firefly luiferse protein produt is lrger. However, wild-type hd mrked effet on firefly luiferse expression, deresing shrply fter only 8 min (Fig., left pnel), n effet tht n ount for the differene in synthesized protein shown in Figure e,f. Wild-type lso modestly, ut reproduily, delyed the initil prodution of firefly luiferse reltive to (RBD), n effet tht proly reflets slower trnsltion elongtion (Fig., right pnel; summrized in Fig. ). The slowed trnsltion elongtion is onsistent with inhiited eefa GTPse tivity in the Ago eefa omplex, ut the effet ws not lrge enough to ount for the 7% redution in the quntity of synthesized protein (Fig. e,f). We onlude tht slows trnsltion elongtion nd loks protein prodution from its trget mrna. AgoeEFA ttenutes trnsltion elongtion We onsidered possile mehnisms y whih might inhiit protein prodution: it might ttenute trnsltion elongtion, onsistent with the riosome drop-off model proposed for mirna-medited trnsltion repression ; it might inhiit trnsltion termintion; or it might promote destrution of nsent polypeptides during trnsltion, s oserved for mirna-medited repression. The first two mehnisms mke preditions out effets on riosome position. If riosomes drop off the mrna during elongtion, riosoml density should e higher t the end of the ORF nd tper off towrd the end. If trnsltion termintion is inhiited, riosomes should Firefly luiferse units ( 6 ) Renill luiferse units ( ) Loded riosomes trnslte mrnas Aumultion of Renill omplete (± ) Assemly of fully-repressive omplex? Aumultion of firefly omplete (RBD) (WT) Time (min) (RBD) (RBD) nture struturl & moleulr iology dvne online pulition

6 Normlized RNA signl RNse-treted in vitro trnsltion retions Frtionte over surose grdient Isolte riosome-ound RNA; Hyridize to rry Hyridize to rry Used to normlize (ABD) (EBD) (RBD) Normlized RNA signl d 7 6 (ABD) 6 8, Nuleotide Deletion Two repression mehnisms: Firefly luiferse ORF (EBD) (RBD) Nture Ameri, In. All rights reserved. 6 8,,,,6 Nuleotide Firefly luiferse ORF Figure Humn ttenutes trnsltion elongtion. () Riosoml footprinting protool. In vitro trnsltion retion experiments were onduted with rdioleled firefly luiferse mrna hroring PBEs, nd retions were quenhed with yloheximide nd treted with RNse. RNA from monoriosome frtions ws olleted nd hyridized to spotted rry with oligonuleotides omplementry to firefly mrna. Signls from monoriosome-ound RNA frgments were normlized with input RNA frgments. () ffets riosome position., (ABD), (EBD) or (RBD) were dded to retiuloyte lyste, nd riosoml footprinting ws rried out s digrmmed in pnel. At the end of the ORF, riosoml density is equivlent for ll smples, level tht ontinues ross the ORF when (ABD, EBD nd RBD) mutnts re dded. With wild-type, riosoml density peks t ~ nt nd dereses to level lower thn tht seen with mutnts. Therefore, trnsltion is ttenuted during elongtion. Note tht smples with wild-type hd more input RNA signl, owing to stilized mrna (Supplementry Fig. 6), whih dmpens the signl oserved for riosoml footprints, one normlized. Error rs represent s.d. (n = ). () As in pnel, ut footprinting ws rried out with mutnt firefly luiferse mrna with ~ nuleotides removed fter the strt odon (grey). As ove, wild-type used riosomes to umulte over the first ~ nt. Riosoml density gin dropped fter this site of umultion to level lower thn tht seen with the mutnts, whih ll hd equivlent riosoml density ross the ORF. (d) Model for how the PUFAgoeEFA omplex ttenutes trnsltion elongtion. See text for explntion. umulte t the end of the ORF. High-resolution riosoml footprinting ws previously desried t genomi level, nd we modified this pproh for our single mrna reporter (Fig. ). Briefly, in vitro trnsltion retion experiments were rried out with rdioleled firefly luiferse mrna (with PBEs). Retion mixtures ontined, (ABD), (EBD) or (RBD). Trnsltion retion mixtures were quenhed with yloheximide nd treted with RNse One, then frtionted over surose grdient to isolte riosome-ound RNA frgments. These RNA frgments were extrted from the monoriosome pek nd hyridized to spotted rry ontining overlpping DNA oligonuleotides omplementry to the firefly luiferse mrna (Supplementry Fig. 9). RNA signls from monoriosome frtions were normlized to signl from unfrtionted smples to ontrol for hyridiztion effiieny, uridine ontent nd RNse sensitivity nd then plotted ross the mrna (Fig. ). We find it striking tht in the presene of, lrge pek ours ~ nt into the ORF, nd then riosoml footprints re shrply redued ross the reminder of the ORF. The pek oinides with the position t whih nsent polypeptide should emerge from the riosoml exit tunnel (~ residues ). Importntly, the riosoml footprints oserved in smples with (ABD nd EBD) were similr to tht with (RBD). Therefore, the riosoml footprinting profile is only ltered when n form ternry omplex on its trget mrna. To ensure tht the oserved riosoml profile ws independent of nuleotide sequene, we repeted our riosoml footprinting experiment with firefly luiferse mrna lking ~ nuleotides fter the initition odon (inluding the sequene where riosomes mrna STOP S 6S AUG AAAAAA umulted ove). Agin, we oserved deresed riosoml density fter the first ~ nuleotides into the ORF only when wild-type ws dded (Fig. ). We onlude tht the AgoeEFA omplex ttenutes trnsltion elongtion, using riosomes to umulte within the ORF. DISCUSSION Our dt support three min onlusions tht dvne our understnding of how PUF nd Ago proteins regulte trnsltion. First, PUF nd Ago proteins n form onserved omplex ontining the ore trnsltion elongtion ftor, eefa. Seond, the nemtode omplex inhiits eefa GTPse tivity, nd the humn omplex represses trnsltion. Third, the omplex loks elongtion t site ~ nuleotides into the open reding frme, where the nsent peptide is expeted to emerge from the riosoml exit tunnel. Below, we disuss the implitions of these findings nd propose model to desrie our oservtions: tht PUFAgo inhiits eefa GTPse tivity nd loks trnsltion elongtion. A onserved PUFAgoeEFA omplex Studies done prior to this work foused on either PUF or Ago proteins,9. One hint tht the two might work together ws ioinformtis nlysis of humn UTRs, reveling nonrndom proximity of PUF protein inding elements nd mirna inding sites 6. Here, we demonstrte not only tht PUF nd Ago proteins intert ut lso tht the PUFAgo heterodimer ssoites with eefa nd inhiits its GTPse tivity. Our fous in nemtodes ws the FBF- PUF protein PUF eefa Ago dvne online pulition nture struturl & moleulr iology

7 Nture Ameri, In. All rights reserved. nd the CSR- Ago protein, ut we suspet tht other PUFAgo omintions my our, s the nemtode genome enodes PUFs nd 7 Agos. FBF- nd CSR- re divergent memers of their respetive fmilies, yet oth proteins possess signture domins. Moreover, CSR- hs sliing tivity despite its nonnonil funtion in hromosome segregtion 9. One possiility might hve een tht the FBF-CSR- ssoition ws typil. However, in mmmls, humn n ssoite with three AGOs (AGO, AGO nd AGO), suggesting onservtion. We suggest tht multiple PUFAgo omintions exist oth in nemtodes nd mmmls. A question for the future is whether individul PUFs nd Agos ehve differently with respet to their prtiiption in the PUFAgoeEFA omplex. Smll RNAs nnot e essentil for formtion of the PUFAgo eefa omplex. The MID-PIWI domin of Ago is suffiient to form the omplex, nd together with its PUF prtner, the MID-PIWI domin inhiits eefa GTPse tivity. Yet the MID-PIWI domin nnot ind smll RNAs on its own. By ontrst, the PAZ domin of Ago is not required to ssemle the omplex (Fig. ), ut this PAZ domin drives smll RNA loding. Nonetheless, PUFAgoeEFA omplexes formed in vivo ontin full-length Agos nd proly ssoite with smll RNAs. An ttrtive ide is tht the PUFAgoeEFA omplex oordintes PUF nd mirna regultion. The intertion etween the PUFAgo heterodimer nd eefa omplex is reminisent of the originl identifition of Ago in omplex with eif (ref. ), whih is struturlly similr to eefa nd lso possesses GTPse tivity. Moreover, two Drosophil RNAinding proteins, dfxr nd VIG, oordinte with Ago in the ontext of mirisc to repress trget mrnas. A promising possiility is tht Ago proteins n work with other RNA-inding proteins nd other trnsltion ftors to modulte multiple steps of protein synthesis. PUFAgoeEFA regultes trnsltion elongtion Figure d presents model for how the PUFAgoeEFA omplex my ontrol trnsltion elongtion. Centrl to this model is its nhor through the PUFAgo heterodimer to regultory elements in the UTR nd its ssoition with n inhiited ore trnsltion elongtion ftor, eefa. Also entrl to this model is lok within the ORF t position orresponding roughly to where the nsent polypeptide emerges from the riosoml exit tunnel. Our kineti nlysis of protein prodution from -repressed reporter mrna (Fig. ) suggested two phses of trnsltionl inhiition. An initil modest inhiition ws inferred from slower elongtion rte, onsistent with inhiition of eefa GTPse tivity; susequent full repression ws inferred from omplete lok in protein synthesis seen lter. Full repression my require onformtionl hnge in the PUFAgoeEFA omplex, require its modifition, or require reruitment of dditionl ftors. If riosomes do indeed umulte where the nsent polypeptide leves the riosoml exit tunnel, the existene of n dditionl ftor tht reognizes the nsent polypeptide seems likely. The model in Figure d does not inlude ny smll RNA ssoited with Ago, euse our work foused on Ago, PUF nd eefa proteins, not smll RNAs. For exmple, n mirna inding site ws not engineered into the reporter UTRs, nd mirnas were not dded to the retion mixtures. One possiility is tht the PUFAgoeEFA mehnism revels role for Ago tht does not rely on smll RNAs. Perhps more likely is the ide tht smll RNAs influene reruitment, either in n essentil or filitting mnner. Addressing the role of smll RNAs in this mehnism is n ovious next step for future nlyses. Repression t the level of trnsltion initition is emerging s fvored mehnism of trnsltionl ontrol y mirnas 9. We provide two lines of evidene tht the PUFAgoeEFA omplex does not hve mjor effet on trnsltion initition. First, polyriosoml profiles were the sme for -repressed mrna nd ontrol mrna (Supplementry Fig. 8). Seond, the sme initil slope ws oserved in our kineti nlysis of protein prodution y -repressed nd ontrol mrnas (Fig. ). In ddition, repression ourred in the presene of exess mrna p nlog, suggesting tht its repression is exerted fter loding S riosoml suunits. Although we nnot exlude the possiility of minor effet on trnsltion initition, our results strongly point to trnsltionl elongtion s the mjor level t whih the PUFAgoeEFA omplex exerts its repressive effets. How does the proposed PUFAgoeEFA mehnism ompre to estlished mehnisms of regultion t the level of trnsltion elongtion? Perhps the est-known mehnism is tht dopted y the signl reognition prtile (SRP) during synthesis of memrne proteins 6, where trnsltion elongtion is inhiited 7 until SRP doks with the endoplsmi retiulum 6. The PUFAgoeEFA mehnism resemles this se in tht riosomes umulte t similr position within the ORF. However, no role for UTR inding proteins is known for the SRP lok. Reently disovered is the hnrnp E ssoition with eefa to inhiit trnsltion elongtion; however, hnrnp E does not inhiit eefa GTPse tivity ut insted inhiits eefa dissoition from the riosome 8. Another se is mirisc, whih is thought to promote riosoml drop-off from trgeted mrna ut not t given position within the ORF. Therefore no estlished mehnism fits the PUFAgoeEFA model ompletely. Why multiple PUF nd Ago mehnisms? The PUFAgoeEFA mehnism ontrolling trnsltion elongtion (Fig. d) is not the only mehnism used y PUF nd Ago proteins, nd it is perhps not even the primry mehnism. Its reltive importne to other well-estlished mehnisms (for exmple, dedenyltion nd instility, see ove) remins unknown. Why use multiple mehnisms? We do not know, ut we hve three suggestions. One is seurity: if the PUF protein or Ago protein fils to silene trget mrna with one mehnism, the regultor my invoke different mehnism to repress the esped mrna. A seond ide is reversiility: destrution is ded end for n mrna, ut loked elongtion might ensure roust tivtion t lter step. A third ide is suellulr omprtmentliztion: s n mrna emerges from the nuleus, it meets one suellulr environment, ut upon loliztion, for exmple to stress grnules, the mrna enters different environment. Trnsltionl repression in (or loliztion to) distint suellulr loles my differentilly use one mehnism over nother. Critil issues for the future inlude unrveling the iologil funtions, reltionships nd regultion of the vrious PUF nd Ago mehnisms. Methods Methods nd ny ssoited referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Struturl & Moleulr Biology wesite. Aknowledgments We thnk H. Tr (Kyoto University) for providing CSR- nd DRH- ntiodies nd E. Kipreos (University of Georgi) for the CYE- ntiody. We thnk Kimle nd Wikens l memers for disussion; we lso thnk E. Lund nd S. Kennedy for ritil reding of the mnusript nd A. Helsley-Mrhnks nd L. Vnderploeg for help with the mnusript nd figure preprtion. Mss spetrometry ws rried out with support from the Humn Proteomis Progrm t the University of Wisonsin-Mdison. nture struturl & moleulr iology dvne online pulition

8 Nture Ameri, In. All rights reserved. AUTHOR CONTRIBUTIONS K.F. onduted the experiments with the exeption of phylogeneti nlysis (Z.T.C.), luiferse reporter mrna prodution (A.C.) nd sr- mutnt genertion (P.K.-C.). K.F., M.P.W. nd J.K. prepred the mnusript. K.F. is supported y PF--7--DDC from the Amerin Cner Soiety, Z.T.C. y US Ntionl Institutes of Helth (NIH) postdotorl fellowship F GM969, A.C. y NIH trining grnt T GM7 nd n Advned Opportunity Fellowship from the University of Wisonsin- Mdison, M.P.W. y NIH grnts GM89 nd GM9 nd J.K. y NIH grnt GM69. J.K. is n investigtor of the Howrd Hughes Medil Institute. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprints/index.html.. Thompson, B., Wikens, M. & Kimle, J. Trnsltionl ontrol in development. in Trnsltionl Control in Biology nd Mediine (eds. 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She, X., Xu, X., Fedotov, A., Kelly, W.G. & Mine, E.M. Regultion of heterohromtin ssemly on unpired hromosomes during Cenorhditis elegns meiosis y omponents of smll RNA-medited pthwy. PLoS Genet., e6 (9).. Meister, G. et l. Identifition of novel rgonute-ssoited proteins. Curr. Biol., 9 ().. Prmeggini, A. & Snder, G. Properties nd regultion of the GTPse tivities of elongtion ftors Tu nd G, nd of initition ftor. Mol. Cell. Biohem., 98 (98).. Cool, R.H. & Prmeggini, A. Sustitution of histidine-8 nd the GTPse mehnism of elongtion ftor Tu. Biohemistry, 666 (99).. Mili, S. & Steitz, J.A. Evidene for ressoition of RNA-inding proteins fter ell lysis: implitions for the interprettion of immunopreipittion nlyses. RNA, 6969 ().. Wng, X., MLhln, J., Zmore, P.D. & Hll, T.M.T. Modulr reognition of RNA y humn pumilio-homology domin. Cell, (). 6. Zmore, P.D., Willimson, J.R. & Lehmnn, R. The Pumilio protein inds RNA through onserved domin tht defines new lss of RNA-inding proteins. RNA, (997). 7. 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Chkrvrty, I., Bghi, M.K., Roy, R., Bnerjee, A.C. & Gupt, N.K. Protein synthesis in rit retiuloytes. Purifition nd properties of n Mr 8, polypeptide (Co-eIF-A8) with Co-eIF-A tivity. J. Biol. Chem. 6, (98).. Cudy, A.A., Myers, M., Hnnon, G.J. & Hmmond, S.M. Frgile X-relted protein nd VIG ssoite with the RNA interferene mhinery. Genes Dev. 6, 996 (). 6. Wlter, P., Irhimi, I. & Bloel, G. Trnslotion of proteins ross the endoplsmi retiulum. I. Signl reognition protein (SRP) inds to in-vitro-ssemled polysomes synthesizing seretory protein. J. Cell Biol. 9, (98). 7. Wolin, S.L. & Wlter, P. Signl reognition prtile medites trnsient elongtion rrest of preproltin in retiuloyte lyste. J. Cell Biol. 9, 676 (989). 8. Hussey, G.S. et l. Identifition of n mrnp omplex regulting tumorigenesis t the trnsltionl elongtion step. Mol. Cell, 9 (). dvne online pulition nture struturl & moleulr iology

9 Nture Ameri, In. All rights reserved. ONLINE METHODS Additionl methods. Strins, plsmids nd ntiodies re detiled in Supplementry Methods. Also ontined in Supplementry Methods re protools for northern lotting, eletrophoreti moility shift ssys nd mss spetrometry. Protein purifition. All GST protein purifitions were rried out in old lysis uffer A ( mm Tris-HCl, ph 7., mm NH SO, mm NCl, mm EDTA, mm DTT nd mm PMSF, supplemented with Rohe Complete Protese Inhiitor Coktil). Cells were lysed, nd lered lyste ws inuted with glutthione-sephrose (GE Helthre). Protein ws eluted in lysis uffer A with mm redued glutthione. When neessry, GST tgs were removed with U PreSission Protese (GE Helthre). His 6 CSR- ws purified s ove with minor modifitions. Rther thn lysis uffer A, lysis uffer B ( mm NH PO, ph 7., % (v/v) glyerol, mm NH SO, mm NCl, mm DTT nd mm PMSF, supplemented with Rohe Complete Protese Inhiitor Coktil) ws used. Smples were seleted with Ni NTA Agrose (Qigen) nd eluted in lysis uffer B with mm imidzole. Immunopreipittion nd trnsient trnsfetion. Lystes from trnsgeni lines expressing GFPøFBF- nd GFPøtuulin were prepred s ove. Clered lystes were treted with nti-gfp immunopreipittion with or without RNse A. Coimmunopreipitted proteins were nlyzed y western lotting. HEK9T ells were trnsfeted using Lipofetmine (Invitrogen), ording to the mnufturer s instrutions. Cells were then treted with.% (v/v) formldehyde nd quenhed with. M glyine. RIPA uffer ws dded ( mm Tris-HCl, ph 8., mm NCl, % (v/v) NP-,.% (w/v) sodium deoxyholte,.% (w/v) SDS, mm EDTA,. µg RNse A). Smples were immunopreipitted with nti-ha ntiody, wshed in lysis uffer nd nlyzed y western lotting. Immunofluoresene nd fluoresene mirosopy. Adult hermphrodite gonds were extruded in M9 uffer (with.% (v/v) Tween- nd. mm levmisole). Gonds were wshed in PBSTw (PBS with.% (v/v) Tween-) nd fixed in % (v/v) prformldehyde, dissolved in mm K HPO (ph 7.). Smples were wshed in PBSTw, nd ie-old methnol ws dded. Smples were then loked for min t C in PBSTw plus.% (w/v) BSA. Primry ntiodies (nticsr- t :, ntigld- t : or nticye- t :) were dded. Smples were wshed with PBSTw plus.% (w/v) BSA nd inuted with the pproprite seondry ntiodies (: dilution), with DAPI. Smples were wshed in PBSTw plus.% (w/v) BSA nd mounted with Vetshield (Vetor Lortories). Visuliztion ws on Zeiss LSM Met onfol mirosope. GFPøHB ws visulized s ove, ut without ntiody tretments. DAPI stining ws rried out. Smples were exmined on n Axio Imger D.. RNAi. To ondut RNA interferene, L hermphrodite feeding ws done. Briefly, the un-9(ed); xis7[pie- prom:gfpøhb:gld- UTR] nd un-9(ed); xis77[pie- prom:gfpøhb:gld- mutnt UTR] strins were piked to RNAi pltes. Control pltes ontined the Esherihi oli strin HT. Only sterile nimls (euse sr-, drh-, ego- nd ekl- ll result in sterility) were stined with DAPI nd sored. Protein inding ssys. Either ng eh of GSTFBF-, GSTFBF-(FR), petduet-expressed GSTFBF-CSR- omplex or GSTFBF-(FR)CSR- omplex were dded to ng purified EFT- in µl of PBSTw supplemented with mm DTT. Retion mixtures were inuted with or without. mm GTP. Retion mixtures were then dded to glutthione-sephrose, inuted nd wshed. Equivlent mounts of input proteins nd pellets were nlyzed. GST- inding ssys were rried out y dding ng of purified, reominnt protein to µl of retiuloyte lyste. Retion mixtures were inuted with RNse A ( µg ml ), diluted with PBSTw nd dded to glutthione-sephrose. Smples were wshed with PBSTw, nd ound proteins were western lotted s indited. GTPse ssy. ng eh of EFT-, EFT-(H9L), GSTFBF-EFT-, GST FBF-CSR-EFT-, or GSTFBF-(FR)CSR- with EFT- were inuted with µci [γ- P]GTP per fmol GTP. Retion experiments were rried out in GTPse uffer ( mm MES/KOH, ph 7., mm NH Cl, mm KCl nd mm MgCl ). Three -µl liquots were quntitted. To these liquots were dded µl of M perhlori id, µl of. M imidzole (ph with HCl) nd µl of.% (w/v) NMoO. Phosphomolydi id ws extrted in utyl ette nd sintilltion-ounted. In vitro trnsltion. In vitro trnsription retion mixtures were treted with T7 RNA polymerse with the mrna p nlog m 7 G ppp G (New Englnd Biols). For riosoml footprinting nlysis, in vitro trnsriptions were rried out with [α- P]UTP. ng GST, GST-, GST-(ABD), GST-(EBD) nd GST- (RBD) were dded to µl nulese-treted, rit retiuloyte lyste (Promeg). µg nondenylted or µg polydenylted firefly luiferse PBEs mrna ws dded, s ws n mino id mixture (Promeg) nd ng ontrol Renill luiferse mrna. After the retion, three -µl liquots were quntitted using the Dul Luiferse ssy system (Promeg). Smples were verged, nd s.d. re from three iologil replites. Polyriosome nlysis. In vitro trnsltion retion mixtures were treted s ove, nd retions were quenhed with either yloheximide or puromyin. Retion mixtures were then seprted over % liner surose grdient. Frtions were olleted in µl liquots nd used for northern lotting. Pulse-hse nlysis. In vitro trnsltion retion mixtures were treted s ove with modifition. Retion mixtures were pre-inuted, nd then m 7 G ppp G (New Englnd Biols) ws dded to onentrtion of mm. Immeditely, liquots were removed over the time ourse. Riosome footprinting. In vitro trnsltion retion mixtures were prepred s ove; however, only rdioleled firefly luiferse mrna ws inluded. Retion mixtures were quenhed with yloheximide. Smples were then treted with RNse One (Amion).. µl were removed (Input RNA) efore the remining smples were seprted over surose grdient. RNA ws extrted from monoriosome frtions nd hyridized to DNA oligonuleotide rry. Smples were ompred to input RNA. Hyridiztion ws for 6 h t C with wsh in nd. SSC for min t C. Error rs indite s.d. etween three iologil replites. Evolutionry tre nd sequene lignment. Conserved sites were identified using evolutionry tre nlysis 9. Protein sequenes were ligned to humn Pum with ClustlW ( Hetmps depiting normlized onservtion Z-sores were generted using Mtl (Mthworks). Conserved residues were visulized on the sis of the struture of humn Pum (ref. ). 9. Rjgopln, L., Pereir, F.A., Lihtrge, O. & Brownell, W.E. Identifition of funtionlly importnt residues/domins in memrne proteins using n evolutionry pproh oupled with systemti muttionl nlysis. Methods Mol. Biol. 9, 8797 (9).. Gupt, Y.K., Nir, D.T., Whrton, R.P. & Aggrwl, A.K. Strutures of humn Pumilio with nonognte RNAs revel moleulr mehnisms for inding promisuity. Struture 6, 97 (8). doi:.8/nsm. nture struturl & moleulr iology