Yeast Polypeptide Exit Tunnel Ribosomal Proteins L17, L35, and L37 are Necessary to Recruit Late-assembling Factors Required for 27SB Pre-rRNA Processing

Yeas polypep ide exi unnel ibosomal p o eins L17, L35 and L37 a e necessa y o ec ui la e-assembling ac o s equi ed o 27SB p e- RNA p ocessing Michael Gamalinda 1 , Jelena Jako lje ic 1 , Reyes Babiano 2 , Jason Talkish 1 , Jesu ´s de la C uz 2 and John L. Wool o d J 1, * 1 Depa men o Biological Sciences, Ca negie Mellon Uni e si y, Pi sbu gh, PA 15213, USA and 2 Depa amen o de Gene ´ ica, Uni e sidad de Se illa, Se illa, E-41012, Spain Recei ed Augus 13, 2012; Re ised Oc obe 14, 2012; Accep ed No embe 5, 2012 ABSTRACT Ribosome syn hesis in ol es he coo dina ed olding and p ocessing o p e- RNAs wi h assembly o ibo- somal p o eins. In euka yo es, hese e en s a e acili a ed by ans-ac ing ac o s ha p opel ibosome ma u a ion om he nucleolus o he cy o- plasm. Howe e , he e is a gap in unde s anding how ibosomal p o eins con igu e p e- ibosomes in i o o enable p ocessing o occu . He e, we ha e examined he ole o adjacen yeas -p o eins L17, L35 and L37 in olding and p ocessing o p e- RNAs, and binding o o he p o eins wi hin assembling ibo- somes. These h ee essen ial ibosomal p o eins, which su ound he polypep ide exi unnel, a e equi ed o 60S subuni o ma ion as a consequence o hei ole in emo al o he ITS2 space om 27SB p e- RNA. L17-, L35- and L37-deple ed cells exhibi u no e o abe an p e-60S assembly in e medi- a es. Al hough he s uc u e o ITS2 does no appea o be g ossly a ec ed in hei absence, hese h ee ibosomal p o eins a e necessa y o e - icien ec ui men o ac o s equi ed o 27SB p e- RNA p ocessing, namely, Nsa2 and Nog2, which associa e wi h p e-60S ibosomal pa icles con aining 27SB p e- RNAs. Al oge he , hese da a suppo ha L17, L35 and L37 a e speci ically equi ed o a ec ui ing s ep immedia ely p eceding emo al o ITS2. INTRODUCTION Ribosomes a e complex mac omolecula machines ha ca alyze he undamen al p ocess o ansla ion. Assembly o ibosomes is a complica ed mul i-s ep p ocess ha in ol es ansc ip ion, olding, modifica ion and p ocessing o p e- RNAs, as well as he concomi an assembly o ibosomal p o eins ( -p o eins) (1–4). In eu- ka yo es, assembly o bo h 40S and 60S ibosomal subuni s begins in he nucleolus wi h he ansc ip ion o p e-35S and p e-5S RNA p ecu so s by RNA polyme ase I (Pol I) and RNA polyme ase III (Pol III), espec i ely (Figu e 1A). Assembly p oceeds h ough a dynamic flux o p o ein–p o ein, p o ein– RNA and RNA– RNA in e ac ions owa d final ma u a ion in he cy oplasm (1,6–9). Mo e han 200 ans-ac ing assembly ac o s a- cili a e hese emodeling e en s ha igge he successi e nucleoly ic emo al o ansc ibed space sequences and he hie a chical addi ion o -p o eins in o nascen subuni s (6,10) (Figu e 1A and B). Pionee ing in i o econs i u ion s udies o bac e ial 30S subuni s es ablished he hie a chical succession o small subuni -p o ein assembly wi h 16S RNA (11–13). RNA s uc u al p obing showed ha binding o an ini ial subse o -p o eins causes s uc u al e- a angemen s in 16S RNA. These ea angemen s o m binding si es ha allow subsequen associa ion o ano he se o -p o eins and s abilize RNA configu a ions (14–19). Wha eme ged om hese s udies is he p inciple ha ibosome syn hesis is highly coope a i e and occu s in pa allel in e media e pa hways (20–22). Howe e , ibosome assembly in i o is much mo e complex, in ol- ing (i) coo dina ion o ansc ip ion wi h assembly, (ii) emo al o space sequences, (iii) expo o p e- ibosomes om he nucleus o he cy oplasm and (i ) con olled ac ion o assembly ac o s. The disco e y o mo e han 200 ibosome biogenesis ac o s has led o ex ensi e in es iga ions on how hey acili a e p ocessing o p e- RNAs and s uc u al ea angemen s o s ee ma u a ion o assembling p e- ibosomal complexes. Al hough he field has lea ned a g ea deal om s udying hem, ans-ac ing ac o s a e *To whom co espondence should be add essed. Tel: +1 412 268 3193; Fax: +1 412 268 7129; Email: [email p o ec ed] Published online 24 Decembe 2012 Nucleic Acids Resea ch, 2013, Vol. 41, No. 3 1965–1983 doi:10.1093/na /gks1272 ßThe Au ho (s) 2012. Published by Ox o d Uni e si y P ess. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by-nc/3.0/), which pe mi s non-comme cial euse, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Fo comme cial e-use, please con ac [email p o ec ed]. only one pa o he g and design ha is ibosome biogen- esis. Ribosomes a e mul imolecula complexes whe ein -p o eins embellish a co e o RNA; hence, -p o eins likely se e as molecula sca olds necessa y no only o ibosome unc ion bu also assembly. Comp ehensi e s udies o how -p o eins acili a e ibosomal subuni bio- genesis a e g ea ly unde ep esen ed. Recen wo k has iden ified o which s eps in p e- RNA p ocessing di e - en -p o eins a e equi ed, bu i is no clea exac ly how hey pa icipa e in hese s eps (23–35). I is p esumed ha hey do no unc ion di ec ly in p e- RNA p ocessing; -p o eins a e hough o ha e s uc u al a he han en- zyma ic unc ions. The oles o -p o eins in es ablishing p e- ibosomal pa icles and ec ui men o assembly ac o s and o he -p o eins o enable subsequen ma u - a ion a e e en less well s udied. Thus a , examina ion o how -p o eins a ec he composi ion o p e- ibosomes o p opel p e- RNA ma u a ion has only been epo ed o a hand ul o -p o eins om bo h -subuni s (36–39). Hence, mo e s udies o his kind a e needed o ully com- p ehend he assembly p ocess. Ini ial sys ema ic in es iga ion o 60S subuni assembly ca ego ized -p o eins based on hei equi emen o ea ly, in e media e, and la e p e- RNA ma u a ion s eps [(30), unpublished da a]. Following up on Po ¨ll e al. (30), we ecen ly cha ac e ized in mo e de ail a subse o -p o eins ha unc ion ea ly in ibosome assembly, du ing he exonucleoly ic imming o ITS1 sequences in 27SA 3 p e- RNAs (39). In his wo k, we ha e ocused on -p o eins L17, L35 and L37, which had been implica ed in emo al o ITS2, and a e adjacen o each o he in he s uc u e o ma u e 60S subuni s (40–42) (Figu e 2). L17 and L35 a e euka yo ic homologs o bac e ial L22 and L29, espec i ely, whe eas L37 does no ha e a bac e ial homolog. Toge he , hese h ee -p o eins in e ac wi h all six seconda y s uc u e domains o 5.8S/25S RNA, ha ing mul iple con ac si es wi h each domain (40–42). They may he e o e be impo an in p omo ing and s abilizing long- ange in e -domain in e ac ions ha ul ima ely influence he a chi ec u e o assembling ibosomes. Thus, hese -p o eins may help c ea e local o global s uc u al p e- equisi es o p e- RNA p ocessing and associa ion o p o eins. They a e also o in e es because, oge he wi h he nonessen ial -p o ein L26 (43), L17, L35 and L37 a e missing om p e- ibosomes in mu an s unable o p ocess ea ly p e- RNA in e media es (39,44). This is a ibu ed o mis olding o RNA domains o which hey bind, as well as eflec ing he coupling o p e- RNA p ocessing wi h -p o ein assembly. In addi ion, Figu e 1. Ribosome assembly pa hway in Saccha omyces ce e isiae.(A) P e- RNA p ocessing pa hway indica ing exonucleoly ic imming and endonucleoly ic eac ions. No e ha co ansc ip ional p ocessing a he A 3 si e also occu s (5) (no shown). (B) Ma u a ion o p e- ibosomal pa icles om he nucleus o he cy oplasm. 1966 Nucleic Acids Resea ch, 2013, Vol. 41, No. 3 since hese -p o eins lie a ound he im o he nascen polypep ide exi unnel, one migh be able o ela e he cons uc ion o his RNP neighbo hood o in e media e p e- RNA ma u a ion s eps. We wan ed o ake a comp ehensi e look a he unc ion o hese h ee -p o eins and how hey a ec ibosome assembly. We es ed hei ole in ec ui ing assembly ac o s o begin o unde s and how hei absence leads o a dis inc p e- RNA p ocessing de ec . We ha e compa ed he pheno ypes upon deple ing each o hese -p o eins and ound ha hey exhibi e y simila de ec s in biogenesis o 60S subuni s e en hough hey a e no in e dependen o associa ion wi h p e- ibosomal pa icles (p e- RNPs). Ou da a indica e ha hese -p o eins a ec ec ui men o a specific se o assembly ac o s necessa y o 27SB p e- RNA p ocessing, and in hei absence p e- RNPs a e g adually u ned o e . MATERIALS AND METHODS Gene a ion o yeas s ains and in i o deple ion o -p o eins Yeas s ains used in his s udy a e de i a i es o ei he JWY6147 (MATa u a3-52 p1-1 lys2-801 his3-200 leu2-1) o BY4741 (MATa his31 leu20 u a30 me 150) as lis ed in Supplemen a y Table S1. Gene dis- up ions, GAL1 p omo e usions, and 3xHA-, 13xMYC- and TAP- agged genes we e cons uc ed using polyme ase chain eac ion (PCR)-based ch omosomal modifica ions (45,46). Oligonucleo ide p ime s o PCR and plasmids used in his s udy a e a ailable upon eques . Like mos yeas -p o eins, L17, L35 and L37 a e encoded by duplica e (pa alogous) genes (47). These -p o ein pai s ha e highly simila , o iden ical, amino acid sequences (Supplemen a y Figu e S1). Condi ional null mu an s ains o RPL17 (GAL-HA-RPL17;MATa u a3-52 p1-1 lys2-801 his3-200 leu2-1 pl17b:: KANMX6 pl17a::GAL-HA-RPL17A TRP1) and RPL35 (GAL-HA-RPL35;MATa u a3-52 p1-1 lys2-801 his3-200 leu2-1 pl35b::KANMX6 pl35a::GAL-HA- RPL35A TRP1) we e cons uc ed by fi s dis up ing one -p o ein gene copy. The p omo e o he mo e highly ex- p essed copy o each duplica e -p o ein gene (48) ha con e s a s onge pheno ype upon dele ion (da a no shown) was he GAL1 p omo e . Thus, we eplaced he comple e open eading ame (ORF) o RPL17B/ YJL177W o RPL35B/YDL136W wi h he KANMX6 casse e ha con e s esis ance o he d ug gene icin (G418). PCR-gene a ed KANMX6 amplicons om he Figu e 2. Localiza ion o L17, L35, and L37 a ound he polypep ide exi unne (PET). (Top le ) Loca ion o L17, L35, and L37 in he s uc u e o ma u e 60S ibosomal subuni s (PDB: 3U5D and 3U5E) (41). CP deno es he cen al p o ube ance. (Top igh ) The 60S ibosomal subuni s uc u e is iewed om he im o he polypep ide exi unnel looking s aigh h ough he subuni . 5.8S RNA is in black, 25S RNA is in g ey, -p o eins o in e es a e shown as colo ed sphe es. Six -p o eins (L22, L24, L26, L31, L38 and L39) adjacen o L17, L35 and L37 a e nonessen ial. (Bo om, le o igh ) RNA domains wi h which -p o eins L17, L35 and L37 in e ac . 5.8S RNA is shown in black, while domains I, II, III, IV, V and VI o 25S RNA a e shown in yellow, blue, o ange, g een, ed, and pu ple, espec i ely. Nucleic Acids Resea ch, 2013, Vol. 41, No. 3 1967 pFA6a-kanMX6 plasmid we e ans o med in o JWY6147, ans o man s we e sc eened o G418 esis - ance, and eplacemen s we e e ified by genomic PCR. F om hese single gene dis up ion backg ounds, he endogenous p omo e o he o he -p o ein gene copy in i s genomic locus (RPL17A/YKL180W o RPL35A/ YDL191W) was eplaced wi h a GAL1 p omo e casse e oge he wi h he TRP1 selec able ma ke . This casse e also con ains a 3xHA-epi ope o place an HA- ag a he N- e minus o he espec i e -p o eins, o enable hei de ec ion ia wes e n blo ing. PCR modules con- aining GAL1 p omo e sequences we e ans o med in o he single -p o ein gene dele ion s ains; ans o man s we e selec ed o g ow h in medium con aining galac ose bu lacking yp ophan. T p+ ans o man s we e sc eened o co ec in eg a ion o he GAL1 p omo e and he N- e minal HA-epi ope usion by wes e n blo ing wi h an i-HA an ise a. A e nume ous a emp s, c ea ing a ch omosomal usion o he GAL1 p omo e o ei he gene copy o RPL37 p o ed o be unsuccess ul. Fo his eason, an RPL37 condi ional mu an s ain (pGAL-RPL37;MATa his31 leu20 u a30 me 150 pl37a::HIS3MX6 pl37b::KANMX4 pGAL-RPL37A LEU2) de i ed om BY4741 was used o cha ac e iza ion. The pl37 mu an s ain used has bo h ch omosomal copies o RPL37 (RPL37A/YLR185W and RPL37B/YDR500C) dele ed and con ains a plasmid bea ing a condi ional allele o RPL37A, wi hou an in- ame N- e minal HA epi ope usion [p ocedu e desc ibed in (26) and (30)]. C- e minal 3xHA-, 13xMYC- and TAP- usion casse es o agging genes by ans o ma ion we e PCR-amplified om plasmids bea ing he sequences o he ags and a selec able ma ke (45,46). His+ o U a+ ans o man s we e sc eened by immunoblo ing wi h an i-HA, -MYC o an i-TAP an ise a o alida e co ec casse e in eg a ion. Condi ional null mu an s ains we e g own a 30Cin ei he ich o syn he ic minimal medium con aining 2% galac ose o exp ess GAL1 usion genes, o shi ed o medium con aining 2% glucose o ep ess exp ession o he -p o ein gene. Cloning o yeas -p o ein genes in o app op ia e ec o s Unique es ic ion si es we e inco po a ed 800–1000 nu- cleo ides ups eam and 50–100 nucleo ides downs eam o he s a and s op codons o -p o ein genes, espec i ely, ia a wo-s ep PCR eac ion. Plasmids om he Yeas Genomic Tiling Collec ion (Open Biosys ems) ha bea in ac RPL17A,RPL35A and RPL37A ORFs as he empla e and oligonucleo ides used we e designed o con ain palind omic sequences ecognized by es ic ion enzymes ha p oduce s icky ends (oligonucleo ide se- quences a ailable upon eques ). Ups eam and down- s eam PCR oligonucleo ides o ampli y each -p o ein gene con ained es ic ion si es as ollows: BamHI and HinDIII o RPL17A; BamHI and SalI o RPL35A; and, BamHI and EcoRI o RPL37A. Resul ing PCR p oduc s we e diges ed wi h he co esponding es ic ion enzymes and hen liga ed in o ec o s diges ed wi h he same es ic ion enzymes. RPL17A- and RPL35A-con- aining PCR amplicons we e cloned in o pRS315 (LEU2) whe eas he casse e bea ing he RPL37A ORF was cloned in o pRS316 (URA3). Co ec plasmid con- s uc s we e e ified by sequencing. Suc ose g adien cen i uga ion P e- ibosomes, 40S and 60S -subuni s, 80S ibosomes, and poly ibosomes we e ac iona ed using a modified p o ocol de i ed om Deshmukh e al. (49). B iefly, 100 ml o loga i hmically g owing yeas cul u es we e ea ed wi h 5 mg cycloheximide 10 min p io o ha es - ing. Fo y A 254 uni s o whole-cell lysa es we e loaded in o 7–47% (w/ ) suc ose g adien s and subjec ed o cen i u- ga ion a 27 000 pm using a So all AH-629 swinging bucke o o . F ac ions we e collec ed and A 254 was moni- o ed using a Teledyne ISCO Foxy R1 densi y g adien ac ion collec o . S eady-s a e analyses o whole-cell mRNAs, p e- RNA in e media es and ma u e RNAs S eady-s a e le els o p e- RNAs we e assayed by p ime ex ension and no he n hyb idiza ion p o ocols adap ed om Ho sey e al. (50). An oligonucleo ide (51) comple- men a y o a egion immedia ely ups eam o he C 2 clea age si e in ITS2 was used o assay o 50-ends o 27S p e- RNA in e media es by p ime ex ension. The same oligonucleo ide was used o assay o 7S p e- RNA by no he n blo ing. T ansc ip ion o GAL1- d i en RPL17A,RPL35A and RPL37A genes in exp es- si e and ep essi e condi ions was also moni o ed by no he n hyb idiza ion using oligonucleo ides comple- men a y o espec i e mRNAs (oligonucleo ide sequences a ailable upon eques ). U2 snRNA was used as loading con ol in all no he n hyb idiza ion assays. To de ec amoun s o ma u e 25S and 18S RNAs, o al RNA was ex ac ed om cells, sepa a ed on a 1% aga ose gel, and isualized by e hidium b omide s aining. Signal in ensi ies o ma u e RNAs we e quan ified using Image Gauge so wa e (Fujifilm). Kine ic pulse-chase labeling analysis Me abolic pulse-chase expe imen s o assay he kine ics o p e- RNA p ocessing in i o we e ca ied ou as desc ibed in Ho sey e al. (50) wi h he ollowing modifica ions: yeas cul u es a OD 610 = 0.4–0.5 we e pulse-labeled wi h [ 3 H-me hyl]-me hionine o 5 min hen chased wi h an excess o non- adioac i e me hionine. RNA was ex- ac ed om samples collec ed a 0, 2, 5, 10, 20, 40 and 60 min a e chase. Equal coun s we e loaded on o aga ose- o maldehyde gels o each RNA sample om GAL-HA-RPL17 and pGAL-RPL37 s ains g own in galac ose-con aining medium o shi ed o 16–17 h o glucose-con aining medium, espec i ely. A fini y pu ifica ion o assembly in e media es TAP- agged assembly ac o s we e used o a fini y-pu i y p e- ibosomes om whole-cell ex ac s using he 1968 Nucleic Acids Resea ch, 2013, Vol. 41, No. 3 single-s ep a fini y pu ifica ion p o ocol desc ibed by Sahas anaman e al. (44). Sil e s aining and wes e n blo ing P o eins om whole-cell ex ac s o pu ified p e- ibo- somes we e sepa a ed by sodium dodecyl sulpha e- polyac ylamide gel elec opho esis (SDS-PAGE) in 4% o 20% T is–Glycine p ecas gels (In i ogen) a e which a s anda d sil e -s aining p o ocol was ollowed. P o eins on SDS-PAGE p ofiles we e labeled based on comig a ion wi h sil e -s ained p o eins p e iously iden ified by mass spec ome y, dec ease in p o ein band in ensi y when exp ession o he co esponding gene is u ned o , o shi in mobili y o epi ope- agged e sions o he co esponding p o ein. De ec ion o p o eins by wes e n blo ing was ca ied ou using he p o ocol adap ed om Sahas anaman e al. (44). Nog2, which comig a es wi h IgG on 4% o 20% p ecas No ex gels, was assayed using 4% o 12% Bis–T is p ecas gels (In i ogen). Co-immunop ecipi a ion o p e- RNAs wi h GFP- agged -p o eins P e- RNAs copu i ying wi h GFP- agged L17 and L37 we e assayed using he one-s ep GFP-T apÕ_A p o ocol (Ch omo ek) as p e iously desc ibed (43). B iefly, RNA en iched om pu ified p e- ibosomes con aining he GFP- agged p o ein was ex ac ed, and p e- RNAs we e assayed by no he n hyb idiza ion ollowing p ocedu es desc ibed by Venema e al. (52). Au o adiog aphs we e analysed using an FLA-5100 imaging sys em (Fujifilm) a he Biology Se ice (CITIUS) om he Uni e si y o Se ille. Fluo escence mic oscopy Expo o p e- ibosomal pa icles om he nucleolus h ough he nucleoplasm o he cy oplasm was assayed by acking he localiza ion o L25-eGFP o S3-eGFP as p e iously desc ibed (36,53–55). Fo his pu pose, each condi ional mu an s ain was ans o med wi h a plasmid ca ying ei he L25- o S3-eGFP, and Nop1-mRFP (55,56). Cells we e imaged using a Ca l Zeiss LSM-510_META_UV_DuoScan in e ed spec al con ocal mic oscope and analysed using ImageJ (57). Timing o -p o ein assembly was add essed by ans o ming YCplac111-RPL17B-eGFP and YCplac111- RPL37B-eGFP in o pl17 and pl37 null s ains, espec - i ely. Each s ain was hen co ans o med wi h ei he pRS316-GAL-NMD3100 o he pRS316-GAL-NMD3FL plasmid, exp essing a GAL p omo e usion o a dominan nega i e unca ion o a wild- ype allele o he NMD3 gene, espec i ely (58). Images we e acqui ed using a Leica DMR mic oscope equipped wi h a DC came a and p ocessed using Adobe Pho oshop 7.0. In i o ITS2 s uc u e p obing ITS2 seconda y s uc u e was assayed by in i o dime hyl sul a e (DMS) p obing using a p o ocol adap ed om Du ca e al. (59). Condi ional null s ains we e g own in exp essi e and ep essi e media as indica ed abo e, o OD 610 = 0.4–0.5. Ten millili e aliquo s o yeas cul u es we e ea ed wi h 200 ml o a esh dilu ion o DMS (Sigma Ald ich) in 95% e hanol (1:4 / ) o a final concen a ion o 50 mM. T ea ed cells we e incuba ed wi h shaking a 30C o 2 min. Reac ions we e quenched by placing he ubes on ice and adding 5 ml o 0.6 M 2-me cap oe hanol and 5 ml o wa e -sa u a ed isoamyl alcohol. As a con ol o he e ec i eness o he s op eac ion, a con ol sample was included whe e DMS ea men was done a e he addi ion o 2-me cap oe hanol and isoamyl alcohol. Cells we e pelle ed by cen i uga ion a 5000 g o 5 min, and he liquid phase was ca e ully emo ed. Cell pelle s we e suspended in 5 ml 0.6 M 2-me cap oe hanol, and cen i uged again. To al RNA was immedia ely ex- ac ed om he cells. Nucleo ide modifica ions we e assayed by p ime ex ension and gel elec opho esis as desc ibed ea lie , using oligonucleo ides complemen a y o ITS2 (a ailable upon eques ). RESULTS P e ious e idence indica es ha bo h L17 and L35 a e equi ed o p ocessing o he ITS2 space sepa a ing ma u e 5.8S and 25S RNA sequences (30,31,44). The ole o L37 in ibosome assembly has no been p e iously epo ed; howe e , ou ini ial cha ac e iza ion led us o belie e ha L37 is also in ol ed in he same assembly s ep. In cells whe e ansc ip ion o RPL35 is ep essed, hence deple ing he p o ein, ma u e 60S subuni s a e no p oduced as consequence o a block in 27SB p e- RNA p ocessing (31) (Supplemen a y Figu es S2 and S3). Nucleocy oplasmic expo is impai ed when L35 is deple ed (Supplemen a y Figu e S3), and p e-60S iboso- mal pa icles a e g adually u ned o e (31). Fu he mo e, L35 assembles in o he p e-60S ibosomal pa icles con- aining 27SA 2 p e- RNA (66SA 2 assembly in e media es; Figu e 1B). Howe e , despi e i s ole in 27SB p e- RNA p ocessing, no in o ma ion is a ailable on how L35 a ec s he s uc u e o ITS2 o ec ui men o specific assembly ac o s o -p o eins in o p e- RNPs. He e, we s a by filling he gap in ou knowledge o he oles o L17 and L37 in p oduc ion o ma u e -subuni s, p e- RNA p o- cessing, and when hey a e s ably loaded in o p e ibosomes. We hen p oceed o show he e ec s o deple ing L17, L35 o L37 on he s uc u e o ITS2, and he associa ion o ans-ac ing ac o s and o he -p o eins wi h p e- RNPs. Deple ion o -p o eins L17 and L37 is de imen al o cellula g ow h and syn hesis o 60S subuni s To assess he unc ion o he essen ial L17 and L37 -p o eins (48,60) in ibosome biogenesis, we u ilized con- di ional null mu an sys ems (GAL-HA-RPL17 and pGAL-RPL37) whe e he co esponding -p o ein gene is exp essed unde he con ol o he GAL1 p omo e (see ‘Ma e ials and Me hods’ sec ion). These s ains ailed o g ow on glucose-con aining solid medium (Figu e 3A) whe e ansc ip ion o GAL1-d i en -p o ein genes is e- p essed (Supplemen a y Figu e S2), and s opped di iding Nucleic Acids Resea ch, 2013, Vol. 41, No. 3 1969 6–9 h a e shi ing om galac ose o glucose-con aining liquid medium (Figu e 3B). Consis en ly, significan e- duc ion o he o al cellula amoun s o HA-L17 wi hin his ime ame is e iden (Supplemen a y Figu e S2). Plasmids bea ing wild- ype RPL17A and RPL37A alleles complemen ed he g ow h de ec o GAL-HA-RPL17 and pGAL-RPL37 s ains on glucose-con aining solid medium, espec i ely (Figu e 3A). To es he e ec o deple ing each p o ein on p oduc- ion o ibosomes, we assayed le els o ma u e RNAs and ibosomal subuni s. Upon deple ion o ei he L17 o L37, s eady-s a e le els o 25S RNA ela i e o 18S RNA dec eased. In each case, he e ec was apid; he a io d opped wi hin 1.5 h a e shi ing om galac ose o glucose (Figu es 4A and Supplemen a y Figu e S4). Consis en wi h hese obse a ions, amoun s o ee 60S -subuni s we e d ama ically educed ela i e o ee 40S subuni s in each o he wo s ains whe e L17 o L37 we e deple ed (Figu e 4B). In addi ion, hal me poly ibosomes we e p esen upon deple ion o -p o eins, indica i e o a defici in 60S -subuni s. These obse a ions indica e ha L17 and L37 a e specifically equi ed o he p ope o - ma ion o ma u e 60S -subuni s. L17 and L37 a e equi ed o p ocessing o 27SB p e- RNA and o ma ion o s able 66S assembly in e media es To es how hese -p o eins a ec o ma ion o ma u e RNAs, we assayed o he syn hesis and u no e o p e- RNAs by pulse-chase expe imen s using [ 3 H-me hyl]-me hionine in GAL-HA-RPL17 and pGAL-RPL37 s ains. Unde pe missi e condi ions whe e L17 and L37 a e exp essed, 35S p e- RNA was apidly p ocessed in o 27S and 20S p ecu so s, which Figu e 3. Deple ion o ei he L17 o L37 leads o cellula g ow h a es . (A) G ow h on solid medium o each condi ional null mu an s ain ela i e o he wild- ype con ol s ains JWY6147 o BY4741. GAL-HA-RPL17,pGAL-RPL37, wild- ype s ains and condi ional s ains episomally exp ess- ing wild- ype alleles o -p o ein genes we e g own in galac ose-con aining (Gal) liquid medium and dilu ed o an OD 610 o 0.5. Ten mic oli e s o 10- o 100 000- old se ial dilu ions we e spo ed on galac ose-con aining (Gal) and glucose-con aining (Glu) solid medium, and hen incuba ed a 30C. (B) Loga i hmically g owing GAL-HA-RPL17 and pGAL-RPL37 cells we e e ained in galac ose-con aining media o ans e ed o glucose-con aining liquid media a 30C. S ains we e dilu ed as necessa y o main ain loga i hmic g ow h, and cell densi ies (OD 610 ) we e con inu- ously measu ed. 1970 Nucleic Acids Resea ch, 2013, Vol. 41, No. 3 we e subsequen ly con e ed o ma u e 25S and 18S RNAs, espec i ely (Figu e 5A). Howe e , in ep essi e condi ions whe e L17 and L37 a e deple ed, syn hesis o 25S RNA was inhibi ed ela i e o 18S RNA. Upon deple ion o ei he -p o ein, 27SB p e- RNA pe sis ed e en a e 10 min o chase, indica ing a delay in p ocessing o his p ecu so species. In e es ingly, we obse ed a g adual dec ease in 27SB p e- RNA o unde ec able le els 20 min a e he chase began, sugges ing e ficien u no e o his p ecu so (Figu e 5A). 23S p e- RNA was also de ec ed in each mu an , albei less so in he absence o L37, sugges ing a delay in p ocessing a si es A 0 ,A 1 and A 2 . The abundance o his in e media e, p e- iously ound in many 60S -subuni assembly mu an s (28,53,61), is mos likely a seconda y consequence o ine - ficien ecycling o assembly ac o s equi ed o he ea ly clea age s eps, which ail o be eleased om abe an 66S p e- ibosomes [discussed in (62)]. We also assayed s eady-s a e le els o p e- RNA in e media es in -p o ein deple ed s ains by p ime ex- ension and no he n hyb idiza ion. Upon deple ion o ei he L17 o L37, we ound no di e ences in amoun s o he p ime ex ension s ops a he A 2 and A 3 si es. Howe e , we obse ed a significan accumula ion o he p ime ex ension s op a he B 1S si e and a mo e modes accumula ion a he B 1L si e (Figu e 5B). This migh be a mani es a ion o he as e ela i e a e o u no e o abo i e 66S in e media es con aining 27SB 1L p e- RNA in mu an s lacking L17 o L37 (28). No he n blo analysis showed ha le els o 7S p e- RNAs dec eased in each mu an (Figu e 5C). The e o e, his indica es ha he obse ed inc ease in B 1L and B 1S ends ep esen 27SB p e- RNAs. Finally, consis en wi h p e ious da a, L17-deple ed cells also accumula ed 50 unca ed in e - media es caused by Ra 1 exonucleoly ic diges ion pas he B 1S si e du ing 27SA 3 p e- RNA p ocessing (44) (Figu e 5B). We conclude ha L17 and L37 a e equi ed o 27SB p e- RNA p ocessing a he C 2 si e in ITS2; in hei absence, abo i e in e media es a e o med bu subse- quen ly a ge ed o u no e . In e es ingly, hese obse - a ions a e e y simila o he p e- RNA p ocessing Figu e 4. Cells deple ed o L17 o L37 exhibi a defici o 60S ibosomal subuni s. (A) To al RNA was ex ac ed om GAL-HA-RPL17 and pGAL-RPL37 s ains shi ed o glucose-con aining media a specified ime poin s. Samples we e sepa a ed on aga ose gels, and s ained wi h e hidium b omide. Ra ios o 25S ela i e o 18S ma u e RNA le els o a leas h ee biological eplica es a e shown in he g aph. (B) Polysomes om GAL-HA-RPL17 and pGAL-RPL37 s ains g own in galac ose-con aining media and shi ed o glucose-con aining media o 17 h we e sedimen ed h ough 7 o 47% suc ose g adien s. 40S and 60S -subuni s, 80S ibosomes and poly ibosomes we e moni o ed a A 254 using a Teledyne ISCO Foxy R1 densi y g adien ac ion collec o . A ows indica e hal me s. Nucleic Acids Resea ch, 2013, Vol. 41, No. 3 1971 pheno ype we p e iously obse ed upon deple ion o L35 -p o ein (31). Abe an p e- ibosomes lacking ei he L17 o L37 a e e ained in he nucle(ol)us Ribosome ma u a ion is ini ia ed in he nucleolus, con- inues in he nucleoplasm, and is comple ed ollowing expo o he p e-40S and p e-60S ibosomal pa icles o he cy oplasm. Al hough e ficien nucleocy oplasmic expo o p e-40S pa icles equi es minimal s uc u al e- o ganiza ion, p e-60S ibosomal pa icles unde go s in- gen s uc u al and composi ional ea angemen s p io o expo (6,7,63). To in es iga e he subcellula loca ion o assembly in e media es in he absence o L17 and L37, we acked he localiza ion o GFP- agged L25, a well-es ablished 60S subuni epo e (64). Impo an ly, L25 is success ully assembled in o p e- ibosomes in he absence o L17 o L37 (Figu e 9C), and hus is a alid epo e in hese mu an s. As expec ed o an -p o ein, we obse ed cy oplasmic localiza ion o L25-eGFP in wild- ype cells g own in galac ose-con aining medium (Figu e 6A). Howe e , GAL-HA-RPL17 and pGAL- RPL37 cells shi ed om galac ose- o glucose-con aining medium exhibi ed inc eased in ensi y o L25-eGFP fluo - escence, bu no he 40S epo e S3-eGFP (Figu e 6A). L25-eGFP signal in ei he L17- o L37-deple ed cells la gely colocalizes wi h he nucleola Nop1-mRFP (53) (Figu e 6B). This indica es he ailu e o o m p e-60S ibosomal pa icles compe en o ansi o he nucleo- plasm and subsequen nuclea expo and ma u a ion in he cy oplasm. Consis en wi h his, i has been epo ed ha assembling p e-60S complexes a e eleased o he nucleoplasm upon comple ion o 27SB p e- RNA p ocess- ing (53). We conclude ha upon deple ion o ei he L17 o L37, he in anuclea anspo o p e-60S complexes om he nucleolus o he nucleoplasm is blocked. Mo eo e , pa - icles ha a e eleased om he nucleolus a e no expo ed o he cy oplasm. Two possible scena ios migh accoun o he la e obse a ion. Fi s , in he absence o L17 o L37, 66S p e- RNPs migh a fic o he nucleoplasm bu a e possibly no in a con o ma ion compe en o expo h ough he nuclea po e. Ano he mo e specific scena io Figu e 5. Deple ion o L17 o L37 leads o a de ec in p ocessing o 27SB p e- RNA and i s e en ual u no e . (A) Syn hesis and u no e o p e- RNAs we e assayed by pulse-chase expe imen s. GAL-HA-RPL17 and pGAL-RPL37 cells we e g own in galac ose- o glucose-con aining medium lacking me hionine o 16–17 h. Cells we e pulse-labeled wi h [ 3 H-me hyl]-me hionine o 5 min and chased wi h an excess o non adioac i e me hionine. To al RNA was ex ac ed, and equal numbe s o adioac i e coun s we e loaded on o dena u ing aga ose gels, ans e ed o ni ocel- lulose memb anes, and exposed o X- ay films. (B) To al RNA was ex ac ed om GAL-HA-RPL17 and pGAL-RPL37 s ains g own in galac ose o shi ed o glucose o 16–17 h a 30C. Samples we e subjec ed o p ime ex ension analysis o 50-ends o 27S and 7S p e- RNAs. The as e isk indica es 50- unca ed p e- RNAs o med specifically in he absence o L17. (C) To al RNA was ex ac ed as desc ibed ea lie , and 7S p e- RNAs we e assayed by no he n hyb idiza ion. U2 was p obed o se e as a loading con ol. 1972 Nucleic Acids Resea ch, 2013, Vol. 41, No. 3 is ha L17 and L37 se e as pla o ms o he expo ma- chine y; hence in hei absence, expo is impai ed. Indeed, some p e-60S expo ac o s localize in close p oximi y o he polypep ide exi unnel (65–67). Recen ly, c yo-EM analyses e ealed ha he expo ac o A x1 in e ac s di ec ly wi h -p o eins in his egion, including L19, L25, L26 and L35 (68). L17 and L37 s ably assemble in o 27SB-con aining p e-60S complexes in he nucleolus The iming o assembly o -p o eins wi h nascen ibo- somes has been b oadly ca ego ized in o ea ly and la e ma u a ion s eps by pulse-labeling expe imen s (69). L17 and L37 we e among hose -p o eins ha associa e ea ly wi h assembling ibosomes. We ook o hogonal app oaches o in es iga e in u he de ail he iming o inco po a ion o L17 and L37 in o p e-60S pa icles: (i) de e mining he localiza- ion o he GFP- agged -p o eins in an NMD3100 dominan nega i e mu an de ec i e o nucleocy oplasmic expo o p e-60S ibosomal pa icles (58,70), (ii) assaying p e- RNA in e media es ha copu i y wi h GFP- agged L17 and L37 -p o eins by no he n hyb idiza ion. Figu e 6. Deple ion o L17 and L37 esul s in e en ion o p e- ibosomes in he nucleus. (A)GAL-HA-RPL17 and pGAL-RPL37 cells exp essing ei he he 60S subuni epo e L25-eGFP o he 40S subuni epo e S3-eGFP we e g own in galac ose- o shi ed o glucose-con aining selec i e medium o an OD 610 o 0.4–0.6. Localiza ion o GFP signal in wild ype and mu an cells was de ec ed using a Ca l Zeiss LSM- 510_META_UV_DuoScan in e ed spec al con ocal mic oscope. (B) Colocaliza ion o L25-eGFP wi h he nucleola ma ke Nop1-mRFP in GAL-HA-RPL17 and pGAL-RPL37 cells g own in galac ose-con aining media and shi ed o glucose-con aining media o 16 h. Nucleic Acids Resea ch, 2013, Vol. 41, No. 3 1973 L35 and L37 condi ional mu an s ha p e en 27SB p e- RNA p ocessing. Mo eo e , iden i ying whe e B ac o s in e ac wi h p e- RNAs o p e- RNPs would enable us o u he unde s and how -p o eins oge he wi h cis- and ans-ac ing ac o s wo k oge he o enable p ocessing o 27SB p e- RNA a ITS2. To ully comp e- hend his p ocessing s ep, i is also a big challenge o iden i y he elusi e endonuclease o C 2 clea age o o de e mine whe he his clea age could be a esul o au oca alysis. The a e o u no e o p e- ibosomes in he absence o L17, L35 o L37 is slowe han in -p o ein mu an s a ec ing ea lie s eps in p e- RNA p ocessing P e iously, ou analysis o L7 and L8 mu an s, which a ec an ea ly s ep o p e- RNA p ocessing, showed sub- s an ially apid u no e o p e- RNAs (39). In con as , L17, L35 o L37 mu an s exhibi mo e delayed u no e o unp ocessed 27SB p e- RNA (31) (Figu e 5A). These de- ec i e p e- RNA in e media es a e p esumably a ge ed o deg ada ion by a su eillance machine y (106–108). A his poin , i is no clea how p e- ibosomal pa icles a e de ec ed as being abe an and wha e en s igge u no e in assembly mu an s. Ne e heless, he gene al pic u e ha eme ges is ha g oss mis olding o RNA may lead o a mo e apid u no e o assembly in e medi- a es, as seen in he L7 and L8 mu an s. P e- ibosome com- posi ion is a ec ed o a a g ea e ex en in he L7 o L8 mu an s han in he L17, L35 o L37 mu an s. When L7 o L8 a e deple ed om p e- ibosomes, he associa ion o se e al p o eins is a ec ed: ea ly- and la e-assembling B ac o s, including Nsa2 and Nog2; A 3 ac o s including Nop15 and Cic1 ha bind ITS2, and E b1 and Nop7 ha bind 25S RNA domains I and III, espec i ely; -p o eins L17, L26, L35 and L37 ha p ima ily bind 5.8S/25S RNA domain I, in addi ion o o he RNA domains; and a numbe o -p o eins adjacen o L7 and L8 ha ha bind o 25S domain II and I, espec i ely (39). This sugges s ha L7 and L8 a e necessa y o es ablish he s uc u e o se e al RNA neighbo hoods, nea by o a he away. The uns uc u ed assembly in e media e in he absence o L7 o L8 may signal ins an aneous u no e o p e- RNAs, ia he exosome o he exonuclease Ra 1, which is p esen in p e ibosomes o L7 o L8 mu an s (39). In s a k con as , upon deple ion o L17, L35 o L37, hus a only Nsa2 and Nog2 a e obse ed o be dec eased in p e- ibosomes, whe eas all he o he -p o eins and assembly ac o s ha ail o associa e wi h p e- ibosomes in he absence o ei he L7 o L8 a e s ill p esen . S ikingly, e en adjacen -p o eins in he neighbo hood su ounding he polypep ide exi unnel a e una ec ed in L17, L35 and L37 mu an s. This sugges s ha ewe p e- RNA egions may be mis olded, accoun ing o he slowe u no e o abe an assembly in e media es in L17, L35 o L37 mu an s. In his case, he unp ocessed ITS2 migh be he a ge o u no e by he exosome. Addi ionally, he A 3 ac o Cic1, a known p o easome adap o (109), which emains associa ed wi h ITS2 o un- p ocessed 27SB in e media es in he absence o L17, L35 o L37, migh also signal u no e . Hence, co ec iming o he emo al o ITS2 migh ye be ano he quali y con ol checkpoin o ensu e ha abe an 27SB- con aining p e- RNPs a e elimina ed. SUPPLEMENTARY DATA Supplemen a y Da a a e a ailable a NAR online: Supple- men a y Table 1, Supplemen a y Figu es 1–7, Supplemen- a y Me hod and Supplemen a y Re e ences [111–113]. ACKNOWLEDGEMENTS The au ho s a e g a e ul o he membe s o he Wool o d and de la C uz labo a o ies o s imula ing discussions and c i ical eading o he manusc ip . They a e also hank ul o he gene ous gi s o an ibodies om D s. M. F omon -Racine, F. Lac ou e, P. Linde , D. Gold a b, J. Maddock, M. McAlea , E. Tos a, J. B odsky, J. Wa ne , L. Lindahl, A. Johnson, S. Rospe and K. Siege s. They hank he ollowing people o gene ously p o iding s ains and plasmids: P. Milke ei , E. Hu , M. N. Hall and A. Jacobson. They also hank H. Teng o con ocal mic oscopy aining and he Molecula Biosenso and Imaging Cen e a Ca negie Mellon o use o hei con ocal mic oscope. 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