Saccharomyces cerevisiae Ribosomal Protein L26 Is Not Essential for Ribosome Assembly and ...

14 Pages·2012·3.51 MB·English

Saccharomyces cerevisiae Ribosomal Protein L26 Is Not Essential for
Ribosome Assembly and Function
ReyesBabiano,aMichaelGamalinda,bJohnL.Woolford,Jr.,bandJesúsdelaCruza
DepartamentodeGenética,UniversidaddeSevilla,Seville,Spain,aandDepartmentofBiologicalSciences,CarnegieMellonUniversity,Pittsburgh,Pennsylvania,USAb
D
Ribosomalproteinsplayimportantrolesinribosomebiogenesisandfunction.Here,westudytheevolutionarilyconservedL26
o
inSaccharomycescerevisiae,whichassemblesintopre-60Sribosomalparticlesinthenucle(ol)us.YeastL26isoneofthemany w
n
ribosomalproteinsencodedbytwofunctionalgenes.Wehavedisruptedbothgenes;surprisingly,thegrowthoftheresulting lo
rpl26nullmutantisapparentlyidenticaltothatoftheisogenicwild-typestrain.TheabsenceofL26minimallyalters60Sribo- a
d
somalsubunitbiogenesis.Polysomeanalysisrevealedtheappearanceofhalf-mers.Analysisofpre-rRNAprocessingindicated e
d
thatL26ismainlyrequiredtooptimize27Spre-rRNAmaturation,withoutwhichthereleaseofpre-60Sparticlesfromthenucle- f
r
(ol)usispartiallyimpaired.RibosomeslackingL26exhibitdifferentialreactivitytodimethylsulfateindomainIof25S/5.8S o
m
rRNAsbutapparentlyareabletosupporttranslationinvivowithwild-typeaccuracy.ThebacterialhomologueofyeastL26,L24,
h
isaprimaryrRNAbindingproteinrequiredfor50Sribosomalsubunitassemblyinvitroandinvivo.Ourresultsunderscore tt
p
potentialdifferencesbetweenprokaryoticandeukaryoticribosomeassembly.WediscussthereasonswhyyeastL26playssuch :/
/
anapparentlynonessentialroleinthecell. m
c
b
.
a
Elucidationofthecrystalstructureofbothprokaryoticandeu- Dictyostelium discoideum (62). Nevertheless, assembly maps are sm
karyoticribosomesatatomicresolution(forexamples,seeref- lacking.Theprocessofr-subunitassemblyinvivoisalsopoorly .o
r
erences7,98,and113andreferencestherein)hasconfirmedthat understood.Assemblyinvivooccursconcomitantlywithprocess- g
/
ribosomeassemblymustbeanextremelycomplexprocess.Pio- ing and modification of the pre-rRNAs, which are well-defined o
n
neeringworkofNomuraandNierhausinthe1970sestablished pathways(seeFig.S1inthesupplementalmaterial).Byfar,ribo-
J
thatbacterialribosomalsubunits(r-subunits)couldbereconsti- somebiogenesishasbeenbeststudiedintheyeastSaccharomyces u
tutedinvitrofrommatureribosomalRNAs(rRNAs)andr-pro- cerevisiae.Ribosomesynthesisproceedsviatheformationofpre- ly
2
teins (40, 41, 65, 69, 70). These experiments demonstrated that ribosomalintermediatesthatcontainr-proteinsandmanynonri- 7
,
r-subunits assemble in a cooperative and hierarchical manner bosomal proteins, so-called ribosome assembly or trans-acting 2
throughreconstitutionintermediates(forreviews,seereferences factors,whichlikelyprovidethisprocesswiththenecessaryspeed, 01
46and93).Hence,ther-proteinswereclassifiedasprimarybind- accuracy,anddirectionality(forexamples,seereferences37,71, 6

b
ingorassemblyinitiatorproteins,whichbinddirectlytorRNA, and91;reviewedinreferences31and53).Thepreribosomalin- y

andsecondaryandtertiarybindingproteins,whichrequireprior termediatesaretermed,accordingtotheirpositionintheribo- U
S
bindingofoneormorer-proteins.Morerecentworkhasdemon- someassemblypathway,90Spreribosomalparticles,nuclearand E
stratedthatbacterialribosomeassemblyinvitrooccursviamulti- cytoplasmic43Spreribosomalparticles,andearly,intermediate, /B
pleparallelpathways(68,99,101).Theextenttowhichbacterial late, and cytoplasmic pre-60S r-particles (16, 21, 31, 103). The C
T
ribosomeassemblypathwaysinvivoresembletheinvitrorecon- complexityofthedifferentparticlesdecreasesduringtheirmatu- A
stitutionexperimentsremainsunclearforanumberofreasons.(i) ration to r-subunits, while concomitant structural rearrange- .G
Assemblyismuchfasterandmoreefficientinvivo,takinglessthan ments allow the stable incorporation of all r-proteins. Insights E
N
3minduringbacterialexponentialgrowthversusthe30to120 into the approximate timing of association and dissociation of
U
minfortheinvitroreconstitutionexperiments(93).(ii)Assembly some of the protein ribosome biogenesis factors have been ob- N
iscotranscriptionalinvivo(17,77).(iii)Assemblyoccursinvivo tainedbystudyingthecompositionofdistinctpre-60Scomplexes IV
withtheprimarypre-rRNAtranscript,whichcontainsspacerse- purifiedfromwild-typecells(reviewedinreferences31and53) E
R
quences,whileinvitroreconstitutionexperimentsaredonewith
and from mutant strains blocked at early, intermediate, or late S
maturerRNAs.(iv)Ribosomeassemblyinvivorequiresdozensof I
T
trans-actingfactors,suchasexo-andendonucleases,RNAheli- A
R
cases,GTPases,chaperones,andrRNA-andr-protein-modifying
Received25April2012 Returnedformodification21May2012 IA
enzymes(reviewedinreferences13and93).(v)Thecomposition Accepted1June2012
oftheinvivoprecursorparticlesisnotidenticaltoinvitrorecon- Publishedaheadofprint11June2012
stitutionintermediatesbutexhibitssomedegreeofsimilarity(69, AddresscorrespondencetoJohnL.Woolford,Jr.,[email protected],orJesús
93).(vi)Otherdiscrepancieswerefoundbetweeninvivoandin delaCruz,[email protected].
vitroribosomeassembly.Forinstance,r-proteinS15,whichisa WededicatethisarticletothememoryofPierreThuriaux.
primarybindingproteinrequiredforbindingoffourother30S R.B.andM.G.contributedequallytothiswork.
r-proteinsinreconstitutionassays,isdispensableinvivo(9). Supplementalmaterialforthisarticlemaybefoundathttp://mcb.asm.org/.
Muchlessisknownaboutthedetailsofeukaryoticribosome Copyright©2012,AmericanSocietyforMicrobiology.AllRightsReserved.
assembly.Todate,invitroreconstitutionofbothfunctional40S doi:10.1128/MCB.00539-12
and60Sr-subunitshasbeenachievedonlyinthesocialamoeba
3228 mcb.asm.org MolecularandCellularBiology p.3228–3241 August2012 Volume32 Number16
L26IsDispensableinSaccharomycescerevisiae
nuclear steps of ribosome maturation (for examples, see refer- Pulse-chaselabelingofpre-rRNA.Pulse-chaselabelingofpre-rRNA
ences56and90andreferencestherein).However,thecourseof wasperformedaspreviouslydescribed(52),using100(cid:2)Ciof[5,6-3H]
theassemblyofther-proteinshasnotbeenreportedwithmuch uracil(45to50Ci/mmol;PerkinElmer)per40OD unitsofyeastcells.
600
precision.Low-resolutionpicturesofearlyandlater-proteinas- Cells were first transformed with an empty YCplac33 plasmid (CEN
semblyhavebeenobtainedbymonitoringthekineticsofinvivo URA3)(seeTableS2inthesupplementalmaterial)tomakethempro-
totrophicforuracil.TheyweregrowninliquidSD-Uramediumtoexpo-
incorporation of labeled r-proteins into preribosomal particles
nentialgrowthphase,pulse-labeledfor2min,andchasedfor5,15,30,and
andcytoplasmicr-subunits(54).Morerecentinvestigationshave
60 min with an excess of cold uracil. Total RNA was extracted by the
analyzedtheincorporationofyeast40Sr-proteinsinto90Sand
acid-phenolmethod(3).About20,000cpmperRNAsamplewereana-
43Spreribosomalparticles(30),whichhaveallowedtheidentifi-
lyzedbothin1.2%agarose–6%formaldehydeand7%polyacrylamide–8
cationofsomeprinciplesgoverningtheassemblyofthe40Sr-sub- D
Mureagels.RNAthenwastransferredtoanylonmembraneandvisual-
o
unitsandtheestablishmentofacertainparallelismbetweenthein izedbyfluorography(52). w
vivoassemblyof40Sr-subunitsandtheinvitroreconstitutiondata Northernhybridizationandprimerextensionanalyses.Steady-state n
lo
ofbacterial30Sr-subunits(30).However,therelativetimingof levelsofpre-rRNAswereassessedbyNorthernandprimerextensionanal- a
theassemblyofonlyafew60Sr-proteinshasbeeninvestigated(4, ysesasdescribedpreviously(107).Inallexperiments,RNAwasextracted d
e
45,84,87,90,112). fromsamplescorrespondingto10OD unitsofexponentiallygrown d
Wewanttounderstandthecontributionof60Sr-proteinsto cells.EqualamountsoftotalRNA(5(cid:2)g6)00wereloadedongelsorusedfor fr
o
ribosomebiogenesis,specificallytheroleoftheseproteinsindriv- primerextensionreactions.Specificoligonucleotides,whosesequences m
ingtheformationand/orrearrangementsofpreribosomalparti- arelistedinTableS3inthesupplementalmaterial,were5=endlabeled h
cles. Previously, a systematic analysis of the role of 26 essential with [(cid:3)-32P]ATP and used as probes. To analyze mRNAs, double- ttp
strandedDNAprobesweregeneratedbyrandomprimingofaPCRfrag- :
yeast60Sr-proteinsinpre-rRNAprocessingandnucleocytoplas- mentoftheRPL26AandRPL26B,RPL35AandRPL35B,andADH1genes //m
mictransportofpre-60Sr-particleswasperformed(78).Before with[32P]dCTP.Thesequencesoftheoligonucleotidesusedasprimers c
thisanalysis,thecontributiontoribosomebiogenesisofonlyafew b
forthePCRsarealsolistedinTableS3. .
a
60Sr-proteinswasstudiedinsomedetail(forexamples,seerefer- Fluorescencemicroscopy.Totestpreribosomalparticleexport,the s
ences4,18,19,45,63,84,85,87,and106).Inthisreport,wehave appropriatestrainswerecotransformedwithpRS316plasmidconstructs m
.
undertakenthefunctionalanalysisofyeastL26inthebiogenesis (giftsfromJ.Bassler),whichexpressthenucleolarmarkermRFP-Nop1 o
r
andfunctionofribosomeswhichremaineduncharacterized.Eu- andeithertheL25-enhancedgreenfluorescentprotein(L25-eGFP)orthe g
/

karyoticL26isaconservedproteinthatsharesnotablesequence S3-eGFPreporter(105).Severaltransformantsthenweregrowntomid- o
n
and structure identity with archaeal and eubacterial L24. L24 is log phase in selective SD liquid medium, washed, and resuspended in J
oneoftwoinitiatorr-proteinsforassemblyof50Sr-subunitsin steriledistilledwater.ToaddresstheassemblypositionofL26,therpl26 u
null strain was cotransformed with YCplac111-RPL26A-eGFP and the ly
vitro(69).OurresultsclearlyshowthatyeastL26assemblesinthe
pRS316-GAL-NMD3(cid:4)100orthepRS316-GAL-NMD3FLplasmid(gifts 2
nucle(ol)uswithintheearliestassemblyintermediatesbutmakes 7
fromA.Jacobson).Theseplasmidsexpressadominant-negativetrunca- ,
veryminorcontributionstothebiogenesis,structure,andfunc- 2
tionorawild-typealleleoftheNMD3gene,respectively,underthecon- 0
tionof60Sr-subunits.Consequently,L26isapparentlydispens- trol of an inducible GAL promoter (6). Transformants were grown in 1
6
ableforcellgrowthunderstandardlaboratoryconditions. selectiveSRafmediumandshiftedtoselectiveSGalmediumtoinducethe b
expressionoftheNmd3proteins.ImageacquisitionwasdoneinaLeica y

U
MATERIALSANDMETHODS DMRmicroscopeequippedwithadifferentialcontrast(DC)camera.Dig-
S
Strainsandmicrobiologicalmethods.Theyeaststrainsusedinthisstudy italimageswereprocessedwithAdobePhotoshop7.0. E
arelistedinTableS1inthesupplementalmaterial.StrainsRBY272and Preparation of ribosome particles. Ribosomes were obtained as /B
previously described (83). Briefly, 200 ml of wild-type or rpl26 null C
RBY274arehaploidsegregantsofY25253andY24664(Euroscarf),re-
spectively.StrainRBY276isahaploidsegregantderivedfromcrossing cellswasgrowninYPDtoanOD600of0.8andconcentratedin500(cid:2)l TA
RBY272 and RBY275, which is another segregant of Y25253. Deletion ofice-coldbuffer1(10mMTris-HCl,pH7.4;20mMKCl;12.5mM .G
disruption,GALconstructs,andC-terminalTAPorMyctaggingatthe MgCl2; 5 mM 2-mercaptoethanol) containing a protease inhibitor E
genomiclocioftheyeastJWY6147wereperformedasdescribedprevi- cocktail.Cellsweredisruptedbyvigorousshakingwithglassbeadsina N
ously(39,61,78,81,110). Fastprep-24 homogenizer at 4°C. An S30 fraction was obtained by U
Growthandhandlingofyeaststrainsandpreparationofstandard centrifugingtheextractat13,000rpmfor20minat4°CinanEppen- N
I
mediawereperformedbyestablishedprocedures(47).Richmedium dorfmicrocentrifuge.RibosomalparticleswerepreparedfromtheS30 V
E
(1%yeastextract,2%peptone;YP)orsynthetic(S)minimalmedium fractionbycentrifugationinaBeckman-CoulterOptimaMaxusinga R
(0.15% yeast nitrogen base, 0.5% ammonium sulfate) were supple- TL110 rotor at 90,000 rpm for 90 min at 4°C. The particles were S
mented with the appropriate amino acids and bases as nutritional washedbycentrifugationina20to40%discontinuoussucrosegradi- IT
requirementsandwith2%galactose(YPGalandSGal,respectively), entinbuffer2(20mMTris-HCl,pH7.4;500mMammoniumacetate; A
2%glucose(YP-dextrose[YPD]andSD,respectively),or2%raffinose 100mMMgCl2;5mM2-mercaptoethanol)andstoredat(cid:5)80°Cinthe RIA
(SRaf).Unlessotherwiseindicated,yeastcellsweregrownat30°Ctoan samebuffer.RibosomalproteinswereseparatedinNuPAGE-SDSwith
opticaldensityat600nm(OD )ofabout0.8.Allsolidmediacon- 4to12%gradientpolyacrylamidegels(Invitrogen),andtheindicated
600
tained2%agar.Low-pHmethyleneblueplateswerepreparedaccord- proteinswereassayedbyWesternblotting.
ingtoastandardprocedure(47). Translational in vivo assays. (i) The sensitivity to different drugs
Plasmids.AllrecombinantDNAtechniqueswereperformedaccord- impairing translation was tested as follows. Wild-type and rpl26 null
ingtoestablishedproceduresusingEscherichiacoliDH5(cid:1)forcloningand strainsweregrowninYPDmediumtoanOD of0.8anddilutedtoan
600
plasmidpropagation(88).Theplasmidsusedinthisstudyaredescribed OD of0.05.Aseriesof10-folddilutionswasdoneforeachstrain,and
600
listedinTableS2inthesupplementalmaterial. 5-(cid:2)ldropswerespottedonYPDplatescontainingthefollowingantibiot-
Sucrosegradientcentrifugation.Polysomepreparationsandanalyses icsattheconcentrationsspecified:anisomycin(15(cid:2)g/ml),azetidine-2-
wereperformedaspreviouslydescribed(52)usinganISCOUA-6system carboxylicacid(AZC;0.5mg/ml),cycloheximide(0.1(cid:2)g/ml),hygromy-
equippedtocontinuouslymonitortheA . cinB(20(cid:2)g/ml),paramomycin(2.5mg/ml),andneomycin(5mg/ml).
254
August2012 Volume32 Number16 mcb.asm.org 3229
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3230 mcb.asm.org MolecularandCellularBiology
L26IsDispensableinSaccharomycescerevisiae
Plateswereincubatedat30°Cfor3to4days.(ii)Thekillervirusassaywas totalcellextractswereobtainedbycentrifugationinamicrocentrifugeat
carriedoutexactlyasdescribedinreference47.Briefly,yeastcolonieswere themaximumspeed(ca.16,100(cid:8)g)for15minat4°C.Eachsupernatant
replicaplatedtolow-pHmethyleneblueplatesfreshlyinoculatedwith0.2 obtainedwasmixedwith40(cid:2)lofGFP-Trap_Abeads,previouslyequili-
mlofanovernightcultureofthesensitive5X47strainandincubatedfor4 bratedwiththesamebuffer,andincubatedfor2hat4°Cwithend-over-
days at 16 or 20°C. The killer activity was scored as a zone of growth endtuberotation.Afterincubation,thebeadswereextensivelywashed3
inhibitionaroundthekillercolonies.(iii)Tomonitorprogrammed(cid:6)1 timeswith5mlofthesamebufferat4°Candfinallycollected.RNAwas
and(cid:5)1frameshifting(PRF),plasmidsp0-LacZ,p-1-LacZ,andp(cid:6)1-LacZ extractedfromthebeadsandtotalcellextractsaspreviouslydescribed(4,
(giftsfromJ.D.Dinman)wereusedexactlyasdescribedpreviously(22, 20)andthenanalyzedbyNorthernblottingasdescribedabove.
23).Plasmidp0-LacZcarriesalacZgeneexpressedfromaPGK1pro- Antibodies. The following primary antibodies were used: mouse
moter. Plasmid p-1-LacZ carries an L-A-derived (cid:5)1 r-frameshift se- monoclonal antihemagglutinin (anti-HA) 12CA5, mouse monoclonal
quenceinsertedinthelacZgene.Plasmidp(cid:6)1-LacZcarriesaTy1-derived anti-Myc(DevelopmentalStudiesHybridomaBank),rabbitpolyclonal D
(cid:6)1r-frameshiftsequenceinsertedinthelacZgene.Wild-typeandrpl26 anti-Has1(agiftfromP.Linder)(28),rabbitpolyclonalanti-Nip7(agift ow
nullcellsweretransformedwiththeseplasmidsandanalyzedfor(cid:7)-galac- fromD.Goldfarb)(117),rabbitpolyclonalanti-Nog1(agiftfromJ.Mad- n
tosidaseactivity(59).TheefficiencyofPRFcorrelateswiththe(cid:7)-galacto- dock)(32),rabbitpolyclonalanti-Nog2(agiftfromM.Fromont-Racine) lo
a
sidaseactivityproducedbycellsharboringp-1-LacZorp(cid:6)1-LacZrelative (90),rabbitpolyclonalanti-Nop7(1),rabbitpolyclonalanti-Nsa2(agift d
e
tothatproducedbycellsharboringp0-LacZ.Allassayswereperformed fromM.Fromont-Racine)(57),rabbitpolyclonalanti-Rlp24(agiftfrom d
foratleastthreeindependenttransformantsofeachplasmidintriplicate. M. Fromont-Racine) (90), rabbit polyclonal anti-Tif6 (a gift from M. f
r
(iv)Tomonitorreadthroughactivity,wild-typeandrpl26nullcellswere Fromont-Racine)(92),mousemonoclonalanti-L3(agiftfromJ.R.War- o
m
transformedwithpUKC815,pUKC817,pUKC818,andpUKC819plas- ner)(109),rabbitpolyclonalanti-L4(agiftfromL.Lindahl),rabbitpoly-

h
mids(giftsfromM.Tuite)(104).pUKC815carriesalacZgeneexpressed clonalanti-L5(19),rabbitpolyclonalanti-L11(67),rabbitpolyclonalanti- t
fromaPGK1promoterandisusedasa(cid:7)-galactosidaseactivitystandard. L17(agiftfromS.Rospert)(79),andrabbitpolyclonalanti-L25(agift tp
:
Expression of (cid:7)-galactosidase activity from pUKC817, pUKC818, and fromK.Siegers)(36).Assecondaryantibodies,goatanti-rabbitoranti- //
m
pUKC819 requires readthrough of the UAA, UAG, and UGA codons, mouse horseradish peroxidase-conjugated (Bio-Rad) or alkaline-phos-
c
respectively.Asdescribedabove,(cid:7)-galactosidaseactivitywasassayedfor phatase-conjugated (Promega) antibodies were used. TAP-tagged b
.
three independent transformants of each plasmid in triplicate. (v) To proteinsweredetectedusingalkalinephosphatase-conjugatedimmuno- a
s
assaythestringencyinstartcodonselection,wild-typeandrpl26nullcells globulins(Pierce).Immunecomplexeswererevealedwithanenhanced m
weretransformedwithp367andp391plasmids(giftsfromA.Hinne- chemiluminescencedetectionkit(Pierce)orusingnitrobluetetrazolium .o
busch)(12).Plasmidp367carriesaHIS4AUG-lacZfusion;p391isaderiv- (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) as the sub- rg
ativeofp367,withUGGasthefirstcodonoftheHIS4-lacZopenreading strates(Promega). /
o
frame(ORF)(HIS4UUG-lacZ).Asdescribedabove,(cid:7)-galactosidaseactiv- Chemicalprobinginvivo.Thesecondarystructureofmature25Sand n

itywasassayedforthreeindependenttransformantsofeachplasmidin 5.8SrRNAwasassayedbyinvivodimethylsulfate(DMS)probingusinga J
u
triplicate. protocoladaptedfromDutcaetal.(26).Briefly,10mlofcellswasgrown ly
Affinitypurificationofpreribosomalparticles.Preribosomalcom- inYPDtoanOD610of0.5andtreatedwith200(cid:2)lofafreshdilutionof 2
plexeswereaffinitypurifiedfromwhole-cellextractswithmagneticDyna- DMS(Sigma-Aldrich)in95%ethanol(1:4,vol/vol)toafinalconcentra- 7
,
beads (Invitrogen), using tandem affinity purification (TAP)-tagged tionof50mM.Treatedcellswereincubatedwithshakingat30°Cfor2 2
0
Nop7assemblyfactorasbait,exactlyasdescribedinpreviousreports(72, min.Reactionswerequenchedbyplacingthetubesoniceandadding5ml 1
87).Purifiedproteinswereprecipitatedin10%trichloroaceticacid,sub- of0.6M2-mercaptoethanoland5mlofwater-saturatedisoamylalcohol. 6

sequentlyresuspendedinSDSLaemmlisamplebuffer,andseparatedby Asacontrolfortheeffectivenessofthestopreaction,acontrolsamplewas b
y
SDS-PAGEin4to20%polyacrylamideNovexgels(Invitrogen).Proteins includedinwhichDMStreatmentwasdoneaftertheadditionof2-mer- U
thenwerevisualizedbysilverstainingorWesternblotting.Westernblot- captoethanolandisoamylalcohol.Cellswerepelletedbycentrifugationat S
tingwasdoneusingastandardprocedure(3).Toprobemultipleproteins 5,000(cid:8)gfor5min,andthentheliquidphasewascarefullyremoved.Cell E
/
fromthesameblot,afterelectroblottingthenitrocellulosemembranewas pelletswereresuspendedin5ml0.6M2-mercaptoethanolandcentri- B
C
cutintosmallersectionsbasedontheknownmobilityoftheproteinstobe fugedagain.TotalRNAwasimmediatelyextractedfromthecells.Nucle-
T
assayed. otidemodificationsintherRNAneighborhoodofL26wereassayedby A
Affinity purification of L26-eGFP protein. GFP-tagged L26A and primerextensionusingoligonucleotidescomplementarytosequencesof .G
L26Bproteinswereprecipitatedbyfollowingtheone-stepGFP-Trap_A the25Sand5.8SrRNAsindomainI(seeTableS3inthesupplemental E
N
procedureslightlymodifiedfromthatsuggestedinthemanufacturer’s material). Reverse transcriptase terminated at the base preceding the

instructions(Chromotek).Briefly,50mlofGFP-taggedoruntaggedneg- modifiednucleotide.Thus,themodifiednucleotidesmigratedoneposi- U
N
ative-controlcellsweregrowninSD-LeumediumtoanOD of0.8, tion higher in the sequencing ladder than in the corresponding DMS
harvested,washedwithcoldwater,andconcentratedin500(cid:2)lo6f00ice-cold lanes. IV
E
lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM CH COOK; 1.5 mM Computationalanalysisofribosomestructure.Atomiccoordinates R
3
MgCl ;1mMdithiothreitol;0.2%TritonX-100)containingaprotease ofthelarger-subunitwereretrievedfromtheProteinDataBank(PDB; S
2 I
inhibitorcocktail(Complete;Roche).Cellsweredisruptedbyvigorous www.rcsb.org)withtheaccessionnumber3OFC(25)forE.coliand3U5D T
A
shakingwithglassbeadsinaFastprep-24(MPBiomedicals)at4°C,and and3U5E(7)forS.cerevisiae.ThemodelswerevisualizedwiththeUCSF
R
I
A
FIG1YeastL26isthestructuralhomologueofbacterialL24.(A)LocalizationofL23(green),L24(red),andL29(blue)inthethree-dimensionalstructureof
theE.coli50Ssubunitandoftheirrespectivehomologues,L25(green),L26(red),andL35(blue),inthethree-dimensionalstructureoftheS.cerevisiae60S
subunit.ThecartoonsweregeneratedwiththeUCSFChimeraprogram,usingtheatomicmodelforthecrystalstructureoftheE.coli70Sribosome(PDBfile
3OFC[25])andtheyeast80Sribosome(PDBfiles3U5Dand3U5E[7]).(B)Close-upviewofthepositionofE.coliL24(left)andyeastL26(right)inthecontext
oftheirbindingsitesintherespectivelargesubunits.OnlyrRNAresiduessituatedatorcloserthan12ÅfromL24orL26areshown(basesandphosphate
backbone).Basenumberingfollowsthe23SrRNAsequencedepositedfortheE.coli50Sr-subunit(PDBfile3OFC[25])andthe25Sand5.8SrRNAsequences
depositedfortheyeast60Sr-subunit(PDBfile3U5D[7]),respectively.Selectedresiduesarelabeledinblack(23Sand25SrRNAs)orinblue(5.8SrRNA).(C)
SecondarystructureofdomainsIfromE.coli23SrRNAandS.cerevisiae25S/5.8SrRNAs.ThestructuresweretakenfromTheComparativeRNAWebSite
(http://www.rna.icmb.utexas.edu/)(11).Yeast5.8SrRNAsequenceishighlightedinyellow.RedcirclesindicaterRNAresiduessituatedcloserthan5ÅfromE.
coliL24oryeastL26proteins.
August2012 Volume32 Number16 mcb.asm.org 3231
Babianoetal.
Chimera program (www.cgl.ucsf.edu/chimera ) 7(6). Secondary struc-
turesofrRNAsfromE.coliandS.cerevisiaeweretakenfromTheCom-
parativeRNAWebSite(http://www.rna.icmb.utexas.edu/)(11).
RESULTS
L26isanevolutionarilyconservedr-protein.Yeastr-proteinL26
is encoded by two paralogous genes (RPL26A and RPL26B), a
featurethatischaracteristicofmostyeastr-proteingenes(111).
Thesegenesproducetwosmallbasicr-proteinsof127aminoac-
ids,L26AandL26B.Ther-proteinsarenearlyidentical,differing D
o
onlyatposition26,glutamineinL26AandglutamicacidinL26B w
(SaccharomycesGenomeDatabase[www.yeastgenome.org]).L26 n
lo
is highly conserved between species, sharing notable sequence a
identity with archaeal and eubacterial L24 (Ribosomal Protein d
e
GeneDatabase[http://ribosome.med.miyazaki-u.ac.jp/];alsosee d

Fig.S2inthesupplementalmaterial).Moreover,prokaryoticL24 fr
o
andeukaryoticL26areRNAbindingproteinsthatoccupysimilar m
locationsinthelarger-subunit.YeastL26islocatednearL35and h
t
L25 in close proximity to the polypeptide exit tunnel; the three tp
:
proteinsareclearlyarrangedinamanneridenticaltotheirbacte- //
m
rial counterparts (Fig. 1A). Most importantly, prokaryotic L24
c
and eukaryotic L26 r-proteins specifically recognize the same b
.
a
structurallyconservedregionindomainIofbacterial23SrRNA s
and eukaryotic 25S/5.8S rRNAs, respectively (Fig. 1B and C). m
FIG2L26assembleswithinthenucle(ol)us.LocalizationofL25-eGFPand .
Thus,itcanbeconcludedthatbacterialL24andeukaryoticL26 L26A-eGFPuponinductionofanNMD3dominant-negativeallele;rpl26null or
areunequivocalfunctionalorthologues. cellsexpressingL25-eGFPorL26A-eGFPweretransformedwiththepRS316- g
/
YeastL26assemblesinthenucle(ol)uswithinearlypreribo- GAL-NMD3(cid:4)100plasmid,andtransformantsweregrowninthepresenceof o
raffinose (SRaf-Leu-Ura). Galactose was then added to fully induce the n
somalparticles.Assemblyofr-subunitsoccursmainlyinthenu- Nmd3(cid:4)100protein.TheGFPsignalwasinspectedbyfluorescencemicroscopy J
cle(ol)us,althoughafew60Sr-proteinsappeartostablyloadonly after24h.Arrowspointtonuclearfluorescence. uly
withcytoplasmicpre-60Sr-particles,suchasL24(54,90),orpref-
2
erentiallywiththem,suchasL10orP0(48,60,84,112).Pulse- 7
,

chase studies have suggested that yeast L26 can assemble at an ribosomesengagedintranslation.Interestingly,27Sand7Spre- 2
0
earlynuclearstageofthe60Sr-subunitmaturationprocess(54); rRNAs were also detected (Fig. 3). In clear contrast, practically 1
6
however,theL26primarysequenceseemstolackapotentialnu- backgroundlevelsweredetectedfor35Sand20Spre-rRNAs.Allof
b
clear localization signal (NLS) (according to the cNLS mapper theseresultsarespecific,sincenoRNAsweredetecteduponaffin- y

program [50]). To explore in which cellular compartment L26 itypurificationfromextractsoftheuntaggedstrain.Similarre- U
S
assembles, we monitored the localization of a functional L26A- sultswereobtainedwhenL26A-eGFPwasusedasthebait(data E
eGFP construct upon induction of the dominant-negative notshown).Takentogether,thesedatastronglysuggestthatL26 /B
NMD3(cid:4)100allele(6).Nmd3istheCrm1-dependentadapterfor stably assembles into early nucle(ol)ar pre-60S r-particles, al- C
T
theexportofpreribosomalparticlesthroughnuclearpores(43). thoughitmightinteractweaklywith90Spreribosomalparticles. A
Thedominant-negativeNmd3(cid:4)100proteintrapspre-60Sr-par- YeastL26iscompletelydispensableforgrowth.Tostudythe .G
ticlesinthenucle(ol)us(43).AsshowninFig.2,theL26A-eGFP role of yeast L26 in ribosome biogenesis and function, we first E
N
constructaccumulatesinthenucleusformostofthecellsexam- analyzed the phenotypic consequences of deleting either the
ineduponoverexpressionoftheNmd3(cid:4)100proteinbutisfound RPL26A or RPL26B gene. As shown in Fig. 4A, rpl26a(cid:4) and UN
in the cytoplasm under noninducible conditions. No change in rpl26b(cid:4)singlemutantsgrowidenticallytothewild-typestrainat IV
the cytoplasmic distribution of L26A-eGFP was observed upon differenttemperatures(16,30,and37°C)onYPDplates.More- E
R
overexpressionofthewild-typeNmd3protein(datanotshown). over,nosignificantdifferenceswerefoundwhendoublingtimes
S
Similar results were obtained when the L25-eGFP reporter was werecalculatedforculturesgrowninliquidYPDorSDmediumat IT
used,whichhasbeenclearlydescribedtoassembleinthenucle- anyofthetemperaturesmentionedabove(datanotshown).To A
(ol)us(forexamples,seereferences4,43,and44). testwhetherL26isrequiredforgrowth,rpl26a(cid:4)andrpl26b(cid:4)hap- RI
A
ToinvestigateinmoredetailthetimingofassemblyofL26,we loid strains were mated and the resulting diploids sporulated.
affinity purified L26B-eGFP-containing complexes using GFP- Mostofthetetradsyieldedfourviablespores.Recombinantdi-
Trapbeads(seeMaterialsandMethods)anddeterminedwhich typeswereobtained,suggestingthatcellscouldgrowwithoutany
pre-rRNA intermediates copurified by Northern blotting. As L26. The absence of both RPL26A and RPL26B was verified by
shown in Fig. 3, and as expected for a 60S r-protein, there was PCR (data not shown). A double disruptant for RPL26A and
significantcopurificationofmature25S,5.8S,and5SrRNAswith RPL26B(herenamedtherpl26nullstrain)grewapparentlyaswell
L26B-eGFP.Moreover,mature18SrRNAwasalsoefficientlyco- asthewild-typestrainonsolidorinliquidmedia(Fig.4Aanddata
purifiedwithL26B-GFP.Aspreviouslydescribed(4),webelieve notshown).Moreover,aGAL::HA-RPL26Arpl26b(cid:4)strain(here
thatthismustreflectthecommonnonspecificassociationof40S namedtheGAL::RPL26strain)grewidenticallyinYPGalandYPD
with60Sr-subunitsin80ScouplesinMg2(cid:6)-containingbuffersor (seeFig.S3inthesupplementalmaterial).Tofurtherascertainthe
3232 mcb.asm.org MolecularandCellularBiology
L26IsDispensableinSaccharomycescerevisiae
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FIG3L26-GFPassociateswithpre-60Sribosomalparticles.GFP-taggedL26wasaffinitypurifiedwithGFP-Trap_Abeadsfromtotalcellularextractsofrpl26 /
o
nullcellsexpressingL26B-eGFP.Wild-typecellswereusedasanuntaggedL26control.RNAwasextractedfromthepelletsobtainedafterpurification(lanesIP) n
orfromanamountoftotalextractscorrespondingto1/100ofthatusedforpurification(lanesT)andwassubjectedtoNorthernanalysisofpre-andmature
J
rRNAs.Probes(inparentheses)aredescribedinFig.S1AandTableS3inthesupplementalmaterial.Signalintensitywasmeasuredbyphosphorimagerscanning; u
values(beloweachIPlane)refertothepercentageofeachRNArecoveredafterpurification. ly

2
7
,

2
0
loss of RPL26 expression in the rpl26 null strain, production of counterpart.BothstrainsdisplayedverysimilarkineticsofrRNA 1
6
RPL26 transcripts was examined by Northern blotting using a synthesis(Fig.6).However,wecouldclearlyobserveamoderate
b
probecommontotheRPL26AandRPL26Bgenes.Asshownin delayinboth35Sand27SBpre-rRNAprocessingfortherpl26null y

Fig. 4B, no specific RPL26 transcript could be detected in rpl26 mutantwithpracticallynoevidentconsequencesinthefinalpro- U
nullcells.Interestingly,theanalysisofsinglerpl26a(cid:4)andrpl26b(cid:4) ductionofboththemature25Sand5.8SrRNA.Thus,theminor SE
strainssuggeststhattheRPL26BgenecontributesmoretoRPL26 deficit in 60S r-subunits in the strain lacking L26 might be the /B
mRNAlevelsthanRPL26A.Altogether,ourresultsclearlydemon- consequenceofaslightdelayin27SBpre-rRNAprocessing. C
T
strate that L26 is completely dispensable for cell growth under Steady-statelevelsofpre-andmaturerRNAswereanalyzedby A
standardlaboratoryconditions.Thisfindingisindependentofthe Northern hybridization and primer extension. Consistent with .G
yeastgeneticbackgroundandclearlynottheconsequenceofthe thepreviousdata,onlymildeffectswereobservedinpre-rRNA E
appearance of spontaneous extragenic suppressors (data not processing,especiallyfortherpl26a(cid:4)andrpl26nullstrains(Fig. N
U
shown). 7).Inthesemutants,therewasaslightaccumulationofboth35S N
TheabsenceofL26leadstoverymilddefectsinribosome and23Spre-rRNAs.The23Sspeciesresultsfrom35Spre-rRNA IV
biogenesis.Thusfar,moststudiedr-proteinscontributetodiffer- cleavageatsiteA withoutpriorcleavageatsitesA ,A ,andA E
3 0 1 2 R
entstepsofribosomebiogenesis(forexamples,seereferences4, (108).Amodestaccumulationof27SA and27SBpre-rRNAswas
2 S
29,and78andreferencestherein).TostudytheroleofyeastL26in alsodetected.Asaconsequence,averyslightdecreaseinthelevels IT
ribosomebiogenesis,wefirstanalyzedpolysomeprofilesfromcell ofmature25Swasfound(Fig.7A;alsoseeTableS4inthesupple- A
R
extractsofthedifferentrpl26deletionstrainsandcomparedthem mental material). Analysis of low-molecular-mass RNA species I
A
tothatfromanisogenicwild-typecounterpart.AsshowninFig.5, showedthatlevelsof7Spre-rRNAsandmature5Sand5.8SrRNAs
therpl26a(cid:4)andrpl26b(cid:4)singlemutantsandtherpl26nullstrain remainedunaffected(Fig.7B).Primerextensionanalysesdemon-
showedaveryminordeficitin60Sr-subunits,asjudgedfromthe strated that the levels of 27SA , 27SA , 27SB , and 27SB pre-
2 3 L S
slightreductionofthelevelsoffree60Srelativeto40Sr-subunits rRNAsmodestlyincreasedintherpl26nullstrain(Fig.7C).Sim-
andtheappearanceofmodesthalf-merpolysomes.Identicalre- ilarprimerextensionresultswereobtaineduponthedepletionof
sults were obtained upon depletion of L26 in the GAL::RPL26 L26(seeFig.S5inthesupplementalmaterial).Weconcludethat
strain(seeFig.S4inthesupplementalmaterial). L26 is not essential for the maturation of rRNAs. Instead, L26
ToclarifywhetherL26isrequiredforpre-rRNAprocessing,we seemstobeneededtooptimizeallofthe27Spre-rRNAprocessing
studiedthekineticsofrRNAproductionby[5,6-3H]uracilpulse- reactions.TheabsenceofL26alsoslightlydelaysprocessingatthe
chaseanalysiswiththerpl26nullstrainanditsisogenicwild-type earlycleavagesitesA toA ,assimilarlyoccursformanymutants
0 2
August2012 Volume32 Number16 mcb.asm.org 3233
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FIG4 L26isnotessentialforgrowth.(A)ThestrainsBY4741(wildtype), b
.
RBY272, a deletant of RPL26A (rpl26a(cid:4)), RBY274, a deletant of RPL26B a
(rpl26b(cid:4)),andRBY276,anrpl26nullstrain(rpl26a(cid:4)rpl26b(cid:4)),weregrownin sm
liquidYPDanddilutedtoanOD of0.05.Serialdilutionswerespottedonto .
YPDplates.Plateswereincubate6d00at30and37°Cfor3daysorat16°Cfor6 FIG5AbsenceofL26resultsinaveryslightdeficitof60Sr-subunits.Poly- or
days.(B)TotalRNAwasextractedfromcellextractofthestrains,separatedby someprofilesareshownforthewild-typeandtheL26-deficientstrainsde- g/
gerenlealencatlryospish.oTrheseiss,atmraensfifeltrerredwtaosacnonylsoencumtievmelybrhaynber,iadnizdesdubwjietchte(cid:1)d-t3o2PN-DorNthA- sacrroiubneddi0n.8F.igT.o4t.aCleelxlstrwacetrsewgreorwenprienpYarPeDd,aatn3d0°1C0aAn2d60haurnvietsstoedfeaatcahnoOnDe6w00eoref on
probesspecificforRPL26,RPL35,andADH1mRNAs.Mature25Sand18S resolvedon7to50%sucrosegradients.TheA254wascontinuouslymonitored. Ju
rRNAs,whichwereusedasmarkerstocheckequalloading,wereprobedwith Sedimentationisfromlefttoright.Thepeaksoffree40Sand60Sr-subunits, ly
(cid:3)-32P-labeledoligonucleotides(seeFig.S1AandTableS3inthesupplemental 80Sfreecouples/monosomes,andpolysomesareindicated.Half-merpoly- 2
material). somesarelabeledbyarrows. 7
,

2
0
1
6
ingenesencoding60Sr-subunitsynthesisfactorsand60Sr-pro- locationofthe60Sr-subunitreporterL25-eGFP(34)inthismu-
b
teins.Thisislikelyduetoinefficientrecyclingoftrans-actingfac- tant and the isogenic wild-type strain. As shown in Fig. 9, L25- y

torsthatcannotefficientlydissociatefromaberrantpre-60Sr-par- eGFPwas,asexpectedforanr-protein,excludedfromthevacuole U
S
ticles(forfurtherdiscussion,seereferences33,85,and108). andfoundpredominantlyinthecytoplasminthewild-typestrain. E
Thecompositionofpre-60SribosomalparticleslackingL26 However, we observed a faint nuclear retention of the fluores- /B
is not significantly affected. To explore the pre-60S ribosomal cencesignaloftheL25-eGFPreporterinabouthalfoftherpl26 C
T
particlesfromanL26-deficientstrain,weaffinitypurifiedNop7- nullcellsexamined.Inmostcells,thefluorescencesignalwasre- A
TAPcomplexesfromGAL::RPL26cellsbeforeorafterdepletionof strictedtothenucleolus,whichwasdetectedwiththenucleolar .G
L26. We then analyzed the protein constituents of the purified marker mRFP-Nop1 (34). No accumulation of nuclear fluores- E
N
complexesbySDS-PAGEandWesternblotting.Nop7-TAPcom- cencewasobservedwhenwestudiedthelocalizationofthe40S
U
plexesareamixtureofearly,intermediate,andlatenuclearpre- r-subunit reporter S3-eGFP (64) in either the wild type or the N
60Sassemblyintermediates(39,71).TheSDS-PAGEprofilesof rpl26nullmutant(Fig.9).Weconcludethatmilddefectsofboth IV
purified pre-60S r-particles from cells depleted of L26 were re- intranuclear and nucleocytoplasmic transport become evident E
R
markablysimilartothosefromcellsexpressingL26.Nochanges when L26 r-protein does not assemble into pre-60S r-particles.
S
wereobservedinthelevelsofthesetofr-proteinsandmostr-sub- These defects are apparently specific, as transport of pre-40S r- IT
unitassemblyfactorsevaluatedbyWesternblotting(Fig.8).How- particlesisunaffectedintheabsenceofL26.Impairedexportof A
R
ever, consistent with the slight delay in processing of late 27S preribosomal particles has been reported in strains mutant for I
A
intermediates observed in an rpl26 null mutant, levels of late- genesencodingr-proteinsandr-subunitbiogenesisproteinsthat
assemblingfactorsNsa2andNog2/Nug2,whicharerequiredfor are not bona fide export factors or adaptors. In such cases, this
ITS2 processing (57, 89), were modestly diminished upon L26 defectsupposedlyarisesfromactivationofanuclearsurveillance
depletion. Thus, the composition of pre-60S r-particles lacking systemthatpreventsexportoftheseparticleswhentheyareaber-
L26isnotsignificantlyaltered. rantormisassembled(discussedinreference102).Thus,pre-60S
Nuclearexportofpre-60Sr-particlesisminimallyimpaired r-particleslackingL26mightbesubstratesofthesenuclearreten-
intheabsenceofL26ribosomalprotein.Tofurthercharacterize tionsystemseventhoughwecouldnotdetectsignificantaltera-
theconsequencesoftheabsenceofL26onribosomebiogenesis, tionsintheircomposition.
we determined whether the rpl26 null mutant was impaired in L26hasaminorroleinribosomestructureandfunction.To
nuclearexportofpre-60Sr-particles.Todothis,weanalyzedthe evaluatechangesintheglobalcompositionofmatureribosomes
3234 mcb.asm.org MolecularandCellularBiology
L26IsDispensableinSaccharomycescerevisiae
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FIG6AbsenceofL26slightlydelaysprocessingof27SBpre-rRNAs.Wild-typeandrpl26nullstrainsweretransformedwiththeCENURA3YCplac33plasmid tp
andthengrownat30°CinSD-UratoanOD600ofaround0.8.Cellswerepulse-labeledfor2minwith[5,6-3H]uracilandthenchasedfor5,15,30,and60min ://
with an excess of unlabeled uracil. Total RNA was extracted, and samples (20,000 cpm per sample) were loaded and separated on a 1.2% agarose–6% m
formaldehydegel(A)ora7%polyacrylamide–8Mureagel(B),transferredtonylonmembranes,andvisualizedbyfluorography.Thepositionsofthedifferent c
b
pre-rRNAsandmaturerRNAsareindicated. .
a
s
m
fromtheL26-deficientstrain,wefirstenrichedribosomesfrom .o
r
wild-typeandrpl26nullcellsbyastandardfractionationprotocol. g
/
r-proteins then were separated by SDS-PAGE and analyzed by o
n
Westernblotting.AsshowninFig.S6inthesupplementalmate-
J
rial, ribosomes from wild-type and rpl26 null cells were clearly u
ly
similar. We next addressed the effect of the loss of L26 on its
2
bindingsiteandtheadjacentrRNAneighborhoodin60Sr-sub- 7
,
units.Todoso,wild-typeandrpl26nullcellsweretreatedwiththe 2
0
RNAmethylatingagentDMS,whichpreferentiallymethylatesad- 1
eninesandcytosinesunlesstheyareprotectedbytheformationof 6

b
basepairs,ternarystructure,orproteinbinding(114).Guanos- y

inesanduracilsarealsomethylatedbyDMSinvivo,albeitrarely U
S
(66).Methylationwasdetectedasstrongstopsinprimerextension
E
reactions(seeMaterialsandMethods).L26interactswithnumer- /B
oussitesindomainIof25S/5.8SrRNA(Fig.1BandC)(7).DMS C
T
probingrevealsthatseveralnucleotidescorrespondingorinclose A
proximitytothebindingregionofL26becomedifferentiallymod- .G
ifiedinitsabsence(Fig.10;alsoseeFig.S7inthesupplemental E
N
material).Asexpected,amajorityofthesesitesbecomemoreac-

U
cessiblewhenL26ismissing.Interestingly,afewunpairednucle-
N
otidesshowdecreasedaccessibilityin60Sr-subunitslackingL26 I
V
(Fig.10;alsoseeFig.S7).Theseresultssuggestthattheabsenceof E
R
L26leadstoclearrRNAconformationalchanges,butthesearenot
S
sufficienttoinducethedestabilizationandlossofotherr-proteins I
T
in60Sr-subunits. A
R
To determine whether these structural differences affect the
I
A
function of L26-deficient ribosomes, we monitored various as-
pectsoftranslationinvivo.First,wemeasuredthehypersensitivity
or resistance to different protein translation inhibitors, which
havebeendescribedasconvenientprobesforchangesinribosome
FIG7AbsenceofL26slightlyalterspre-rRNAprocessing.Thewild-typeand
L26-deficientstrainsdescribedinthelegendtoFig.4weregrowninYPDat function (115). Second, we tested the maintenance of the killer
30°CandharvestedatanOD ofaround0.8.TotalRNAwasextractedand virussystem,whichishighlysensitivetochangesintheconcen-
600
subjectedtoNorthernhybridizationorprimerextension.Probes(inparen- trationoffree60Sr-subunits(74).Third,weevaluatedtransla-
theses)aredescribedinFig.S1AandTableS3inthesupplementalmaterial. tionalaccuracybyquantitativelymonitoringchangesin(cid:5)1and
(A)Northernanalysisofhigh-molecular-masspre-andmaturerRNAs.(B)
(cid:6)1PRF,thereadthroughofnonsensecodons,andthestringency
Northernanalysisoflow-molecular-masspre-andmaturerRNAs.(C)Primer
extensionanalysisof27Spre-rRNAs.ProbefwithinITS2wasused. ofstartcodonselection(23,59,104).Surprisingly,theabsenceof
August2012 Volume32 Number16 mcb.asm.org 3235
Babianoetal.
ture,andfunction.L26anditsbacterialparalogueL24maponto
equivalent positions surrounding the nascent polypeptide exit
tunnel in both eukaryotic and prokaryotic large r-subunits, re-
spectively.
Analysis of an rpl26 null mutant shows that L26 is entirely
dispensableforcellgrowthatdifferenttemperaturesunderstan-
dardlaboratoryconditions.Similarresultshavebeenrecentlyre-
ported by Steffen and coworkers (97). Ten other 60S r-subunit
proteinshavebeendescribedasnotbeingessentialinyeast(5,8,
15,24,35,75,78,80,86,96,97,116).Theabsenceofsomeofthese D
o
nonessentialr-proteinsreducescellgrowthratestodifferentex- w
tents;forexample,eitherL38(96)orL41(116)isalmostdispens- n
lo
ableforgrowth,whileL12ispracticallyessential(8). a
d
Here,wehaveaddressedtheroleofL26r-proteininribosome e
d
biogenesis. The dispensability of L26 denotes that almost wild-
f
typeribosomesarebeingassembledinanL26-deficientstrainun- ro
m
der standard laboratory conditions. Hence, polysome profile,

pulse-chase,Northernblot,primerextension,andpreribosomal h
t
t
compositional analysis clearly indicate that the contribution of p
:
/
L26toribosomebiogenesisisveryminor.IntheabsenceofL26, /m
wecouldobserveonlyamodestdeficitof60Sr-subunits.Thisis c
b
FIG8DepletionofL26doesnotsignificantlyaffectcompositionofpre-60S most likely due to delayed 27SB pre-rRNA processing, which .a
ribosomalparticles.(A)TAP-taggedNop7wasusedtopurifynuclearpre-60S mightbebroughtaboutbymoderatelyreducedlevelsofassembly sm
particlesfromaconditionalGAL::RPL26strain(JWY9634;GAL::HA-RPL26A factorsrequiredforefficientprocessingof27SBpre-rRNAs(i.e., .
rpl26b(cid:4)NOP7-TAP)growninYPGal(Gal)orshiftedtoYPD(Glc).Copuri- Nsa2andNog2)inpre-60Sr-particleslackingL26.Consequently, or
fyingproteinswereseparatedbySDS-PAGEandstainedwithsilver.(B)West- g
ernblottingwasusedtospecificallyassaythepresenceofselectedribosome therelativeamountsof27Spre-rRNAsslightlyincrease,although o/
assemblyfactorsandribosomalproteinsinNop7-TAP-containingpreribo- boththekineticsandthesteady-statelevelsof7Spre-rRNAsre- n

somalparticlesbeforeandafterdepletionofL26. mainunaffected.ThedeficiencyofL26alsoleadstoamilddelayin J
u
pre-rRNA processing at the early cleavage sites A , A , and A ly
0 1 2
withoutconsequencesinthelevelsof20Spre-rRNAandmature 2
L26doesnotresultinchangesin60Sr-subunitsthatsignificantly 7
impactthesetranslationproperties(summarizedinTableS5in 18SrRNA. , 2
thesupplementalmaterial). WehaveanalyzedthestructureofL26-deficientandwild-type 0
1
ribosomesbyDMSprobingexperiments.Ourresultsindicatethat 6
DISCUSSION theabsenceofL26inducessignificantchangesintheconforma- b
y
In this work, we have addressed the role of the evolutionarily tionof25S/5.8SrRNAdomainI.Asexpected,L26-deficientribo-
U
conservedr-proteinL26inyeast60Sr-subunitbiogenesis,struc- somesshowedincreasednucleotidemodificationrelativetowild- S
E
/
B
C
T
A
.
G
E
N

U
N
I
V
E
R
S
I
T
A
R
I
A
FIG9 AbsenceofL26leadstosomenuclearretentionofthe60Sr-subunitreporterL25-eGFP.StrainsBY4741(wildtype)andRBY276(rpl26a(cid:4)rpl26b(cid:4))were
transformedwithplasmidsthatexpressedNop1-mRFPandeitherL25-eGFPorS3-eGFPfromtheircognatepromoters.Cellsweregrownat30°CinSD-Ura.The
subcellularlocalizationoftheGFP-taggedribosomalproteinsandtheNop1-mRFPnucleolarmarkerwereanalyzedbyfluorescencemicroscopy.Arrowspoint
tonucleolarfluorescence.
3236 mcb.asm.org MolecularandCellularBiology
L26IsDispensableinSaccharomycescerevisiae
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FIG10AbsenceofL26induceschangesinthestructureof25S/5.8SrRNAdomainI.(A)InvivoDMSchemicalprobingexperimentoftherRNAstructure V
aroundthebindingsiteofL26.Wild-typeandrpl26nullcellsweretreatedwithDMS.TotalRNAwasextractedfromunmodified((cid:5)DMS)andmodified((cid:6)DMS) E
R
yeastcellsandanalyzedbyprimerextensionusingthe5.8S_3=ENDprimer(seeTableS3inthesupplementalmaterial).U,A,G,andCrepresentdideoxy
S
sequencinglanesdonewiththesameprimer.StopcontrolsindicatetheefficiencyofquenchingreactionsafterDMStreatment.Blackandgreendotsdenote I
T
nucleotideswithstrongerandweakerreactivitytoDMSintherpl26nullstrain,respectively.(B)Secondarystructureofyeast25S/5.8SrRNAdomainI(thelegend A
toFig.1providesfurtherdetails).Redcirclesindicatenucleotidessituatedcloserthan5ÅfromtheyeastL26protein.Nucleotideswithstrongerandweaker R
reactivitytoDMSintherpl26nullstrainarelabeledwithblackorgreenarrowheads,respectively(seeFig.S7inthesupplementalmaterialforfullDMSprobing IA
gels).(C)Close-upviewofthethree-dimensionalarrangementoftheyeast25S/5.8SrRNAdomainI,includingtheribosomalproteinsL17,L35,andL37(red);
L26(red;thesurfaceisalsoshown);andL39(yellow).OnlythebasesidechainsofthenucleotidesundergoingchangesintheDMSreactivityarehighlightedand
labeledinblackorgreen,asdescribedabove.Notethatthestructureisclippedtosimplifythefigure.
typeonesinregionsatornearthebindingsiteofL26.SomerRNA alsoobservedthatafewnucleotidesthatdonotparticipateinbase
residuesboundbyadjacentr-proteinsinmatureribosomesalso pairing showed decreased DMS modification in the absence of
becamemoreaccessibletoDMS.Thisismostlikelynotduetothe L26, suggesting that these nucleotides are now involved in base
mereabsenceoftheser-proteins,sinceL26-deficientribosomes pairingorareprotectedbyanr-proteinincloseproximity.We
remainotherwiseintactrelativetothewildtype.Furthermore,we concludethattheabsenceofassemblyofL26slightlyaffectsthe
August2012 Volume32 Number16 mcb.asm.org 3237