structure BOX/ACA-motif snoRNPs and constitute a common bipartite RNA-binding protein, are ...

21 Pages·1998·3.76 MB·English

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Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative
RNA-binding protein, are present together with Gar1p in all H
BOX/ACA-motif snoRNPs and constitute a common bipartite
structure

N. J. Watkins, A. Gottschalk, G. Neubauer, B. Kastner, P. Fabrizio, M. Mann and R. Luhrmann

RNA 1998 4: 1549-1568
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RNA(1998),4:1549–1568+CambridgeUniversityPress+PrintedintheUSA+
Copyright©1998RNASociety+
Cbf5p, a potential pseudouridine synthase,
and Nhp2p, a putative RNA-binding protein,
are present together with Gar1p in all
H BOX/ACA-motif snoRNPs and constitute
a common bipartite structure
NICHOLAS J. WATKINS,1 ALEXANDER GOTTSCHALK,1 GITTE NEUBAUER,2
BERTHOLD KASTNER,1 PATRIZIAFABRIZIO,1 MATTHIAS MANN,2
and REINHARD LÜHRMANN1
1InstitutfürMolekularbiologieundTumorforschung,Philipps-UniversitätMarburg,
EmilMannkopff-Straße2,D35037Marburg,Germany
2EuropeanMolecularBiologyLaboratory,Meyerhofstrasse1,Postfach102209,69012Heidelberg,Germany
ABSTRACT
The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in pre-
ribosomal RNA(pre-rRNA) processing. The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demon-
strated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine (C) synthesis. To
characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP
complexes by anti-m G-immunoaffinity and Mono-Q chromatography of Saccharomycescerevisiaeextracts. Se-
3
quence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are
commontoboththesnR30andsnR42complexes.Nhp2pisahighlybasicproteinthatbelongstoafamilyofputative
RNA-bindingproteins.Cbf5phasrecentlybeendemonstratedtobeinvolvedinribosomebiogenesisandalsoshows
strikinghomologywithknownprokaryoticCsynthases.ThepresenceofCbf5p,aputativeCsynthaseineachH/ACA
snoRNPsuggeststhatthisclassofRNPsfunctionsasindividualmodificationenzymes.Immunoprecipitationstudies
usingeitheranti-Cbf5pantibodiesorahemagglutinin-taggedNhp2pdemonstratedthatbothproteinsareassociated
withallH/ACA-motifsnoRNPs.InvivodepletionofNhp2presultsinareductioninthesteady-statelevelsofallH/ACA
snoRNAs. Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a
similarbipartitestructurethatweproposetobeamajorstructuraldeterminingprincipleforallH/ACAsnoRNPs.
Keywords: ribosomalRNAprocessing;RNA-bindingproteins;rRNAmodification;Saccharomycescerevisiae;
smallnucleolarRNAs
INTRODUCTION anendonucleaseincleavingthepre-rRNAupstreamof
the 5+8S rRNA (Lygerou et al+, 1996)+ The majority of
The nucleolus contains a multitude of small RNAs
the snoRNAs have been demonstrated to function as
(snoRNAs) that function in ribosome biogenesis+ Sev-
sequence-specific guides in the nucleotide modifica-
eral of these snoRNAs have been shown to be re-
tion of rRNA transcripts+ These snoRNAs are either
quired for pre-rRNA cleavage events (reviewed in
involved in the modification of sugars to create 29-O-
Maxwell&Fournier,1995;Tollervey&Kiss,1997;Smith
methylgroups(Cavailleetal+,1996;Kiss-Laszloetal+,
&Steitz,1997)+RNaseMRPistheonlysnoRNPcom-
1996; Tycowski et al+, 1996) or the isomerization of
plexthathasbeendemonstratedtofunctioninvitroas
uridine to pseudouridine (C) (Ganot et al+, 1997a; Ni
etal+,1997)+IthasalsobeenpostulatedthatsnoRNAs
Reprintrequeststo:ReinhardLührmann,InstitutfürMolekularbi- function as chaperones that help fold the rRNA tran-
ologieundTumorforschung,Philipps-UniversitätMarburg,EmilMann-
scripts, and also in the transport of the mature ribo-
kopff-Straße 2, D35037 Marburg, Germany; e-mail: luehrmann@
imt+uni-marburg+de+ somalsubunitstothecytoplasm(reviewedinBachellerie
1549
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1550 N.J.Watkinsetal.
etal+,1995;Maxwell&Fournier,1995;Steitz&Tycow- cases, has recently been shown to be associated with
ski,1995;Smith&Steitz,1997;Tollervey&Kiss,1997)+ snR30, snR10, and U3 (Ursic et al+, 1997)+
WiththeexceptionoftheRNAmoietyofRNaseMRP The rRNAC synthase has yet to be identified; how-
(7-2 RNA), all known snoRNAs can be classified into ever, it is entirely possible that the protein responsible
one of two categories based on conserved-sequence for the enzymatic activity is an integral component of
motifs+ SnoRNAs contain either the nucleotide box C theH/ACAsnoRNPs+TheprokaryotictRNAandrRNA
and D (C/D) motif or the recently characterized box C synthases do not use a guide RNA to recognize
H/ACA motif (H/ACA) (Balakin et al+, 1996; Ganot specific RNA substrates+ Multiple tRNA C synthases
etal+,1997b)+TheboxC/DclassofsnoRNAsusesbox exist in prokaryotes that recognize either a single site
DorD9,inconjunctionwithlargerRNA-complementary orseveralsiteswithasimilarstructure(Kammenetal+,
sequences, to direct the site-specific 29-O-methylation 1988; Nurse et al+, 1995; Wrzesinski et al+, 1995a,b;
ofrRNA(Cavailleetal+,1996;Kiss-Laszloetal+,1996; Simos et al+, 1996; Lecointe at al+, 1998)+ Therefore,
Tycowski et al+, 1996)+ The H/ACA snoRNAs snR30 the use of a guide RNAto direct rRNAC synthesis in
andsnR10havebeendemonstratedtoberequiredfor eukaryotes is quite unique (Ganot et al+, 1997a; Ni
the 18S rRNAcleavage events (Tollervey, 1987; Mor- et al+, 1997)+ It has recently been demonstrated that
rissey &Tollervey, 1993)+ However, the majority of the Gar1p, a protein common to all H/ACA snoRNPs, is
H/ACAsnoRNAshaverecentlybeenimplicatedtofunc- required for C formation in yeast (Bousquet-Antonelli
tion as guide RNAs in the site-specific pseudouridyl- et al+, 1997)+ It is, however, highly unlikely that this
ationofrRNA(Ganotetal+,1997a;Nietal+,1997)+This proteinisthemodificationenzyme,becauseGar1phas
classofsnoRNAhasbeendemonstratedtopossessa little homology to known C synthases and is therefore
commonhairpin-hinge-hairpin-tailstructurethatincludes postulated to function as an accessory protein in this
twoevolutionarilyconservedsequenceelementstermed event (Bousquet-Antonelli et al+, 1997)+ One possible
the H box and the ACA motif (Ganot et al+, 1997b)+ A candidate for the C synthase is Cbf5 (mammalian ho-
bulge structure (C pocket) within one or both of the mologue NAP57; Meier & Blobel+, 1994), a nucleolar
stems of the snoRNA base pairs with the rRNA on protein involved in ribosome biogenesis that shows a
eithersideofthesubstrateuridine,thustargetingitfor remarkably high degree of sequence similarity with
modification+ Either an H box or ACA motif is located knownCsynthases(Koonin,1996;Cadwelletal+,1997)+
between 14 and 16 nt downstream of the modification Indeed, while we were preparing this article, a paper
site in the C pocket (Ganot et al+, 1997a; Ni et al+, describing Cbf5p as an integral H/ACA snoRNP pro-
1997)+ Since both the H box and the ACA motif are tein involved in snoRNA biogenesis, 18S rRNA pro-
proposed to be protein-binding sites it is thought that cessing, and C synthesis was published (Lafontaine
polypeptidesinteractingwiththeseconservedmotifsare et al+, 1998)+
intimatelyinvolvedinCsynthesis(Ganotetal+,1997a)+ The recent demonstration of the isolation of yeast
The conserved nature of the hairpin-hinge-hairpin- snRNP complexes coupled with the advances in sen-
tailstructureoftheH/ACAsnoRNAswouldsuggestthe sitivity in protein sequencing has resulted in the de-
possibility that this motif could serve as a binding site tailed biochemical and genetic characterization of the
for common proteins+ A similar scenario is observed yeast U1 snRNP (Neubauer et al+, 1997; Gottschalk
with the spliceosomal snRNPs, where a set of eight et al+, 1998)+ Here we describe the application of such
commonproteinsassociatewiththecoreSmsequence techniquestotheisolationandcharacterizationofyeast
(reviewed in Will & Lührmann, 1997)+ One protein, H/ACAsnoRNPs+ThesnR30andsnR42snoRNPswere
Gar1p,wasdemonstratedtobecommontoallH/ACA purifiedbyanti-m G-immunoaffinityandMono-Qchro-
3
snoRNPs by co-immunoprecipitation experiments (Gi- matography and their individual protein components
rard et al+, 1992; Balakin et al+, 1996; Ganot et al+, identifiedbymassspectrometrypeptideanalysis+Gar1p,
1997b)+ The snR30 (H/ACA) snoRNP has been puri- Nhp2p,andCbf5pwerefoundtobepresentinboththe
fied by a combination of anti-m G-immunoaffinity and snR30andsnR42snoRNPs+Thesethreeproteinswere
3
Mono-Q anion exchange chromatography+ This puri- also demonstrated to be associated with all known
fied complex contains at least seven proteins, four of H/ACA snoRNAs+ In vivo depletion studies demon-
which (the 65-, 25-(Gar1p), 23-, and 10-kD proteins) strated that Nhp2p is also required to maintain the
arecandidatesforastableboundcoreastheyremain steady-state levels of H/ACA snoRNAs+ Electron mi-
associated with the RNAin cesium sulphate gradients croscopy analysis of negatively stained core H/ACA
(Lübben et al+, 1995)+ Very few H/ACA snoRNP spe- snoRNPsrevealedthatacommonconservedbipartite
cificproteinshavebeenidentified+Theonlycharacter- structure defines this class of RNP+ These data pro-
ized H/ACA snoRNP specific protein is Ssb1p, which vide the first major insight into the composition and
has been reported to be present in the snR10 and structureofH/ACAsnoRNPs,andsuggestthattherRNA
snR11 snoRNP complexes (Clark et al+, 1990)+ Inter- C synthase is an integral component of these RNP
estingly, Sen1p, a member of the superfamily I heli- complexes+
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CommonH/ACAmotifsnoRNPproteins 1551
RESULTS withapparentmolecularmassesof25,27,and65kD,
suggesting that they may constitute a common core
proteinelement+ThemajorityofsnR42RNAispresent
Purified snR42 and snR30 snoRNPs
in fraction 11; however fractions 17, 18, 23, and 24
share three common proteins
probably contain free snR42 RNA that has been dis-
We have previously demonstrated that the snR30 sociated from its protein components+
snoRNPcanbeisolatedfromyeastwhole-cellextracts
byanti-m G-immunoaffinityandMono-Qchromatogra-
3 Identification of snR42 and snR30 snoRNP
phy (Lübben et al+, 1995)+ Here we have modified this
proteins by mass spectrometry
approach to enable the isolation of sufficient material
for mass spectrometry peptide analysis and to allow Thenextstepwastoidentifytheproteincomponentsof
the purification of other H/ACA box snoRNP com- thesetwopurifiedsnoRNPcomplexes+Theinitialfocus
plexes+ Briefly, total snRNPs and snoRNPs from a wouldbedirectedtowardsthe25-,27-,and64-kDpro-
whole-cellSaccharomycescerevisiaeextractwereen- teinsastheseappeartobeprimecandidatesforcom-
richedbyanti-m G-immunoaffinitychromatography+To mon core H/ACA snoRNP proteins+ The snR30 and
3
simplifythesubsequentchromatographicsteps,theU1 snR42snoRNPswereisolatedfromextractderivedfrom
snRNP,whichcontainsahistidine-taggedU170-kDpro- 100Lofyeastculturetogeneratesufficientmaterialfor
tein, was depleted by passing the extract over a Ni21- massspectrometryanalysis+WhenthepurifiedsnR42
nitrilotriaceticacid–agarosecolumn(Lübbenetal+,1995; proteins were stained with Coomassie, only the 25-,
Neubauer et al+, 1997)+ The flow-through of such a 27-,and65-kDproteinswerevisible+Itispossiblethat
column was then subjected to Mono-Q chromatogra- the 80-kD band is a snR42-specific protein that does
phy+ Elution was performed with a three-step gradient notstainwellwithCoomassie+TheCoomassiestained
from 50 mM to 1M KCl at pH 7+ Three RNP peaks bandsforboththesnR30andsnR42snoRNPproteins
absorbingat260nmand280nmwereobservedelut- wereexcisedfromapolyacrylamidegel,characterized
ing from the column at approximately 550 mM, by mass spectrometry, and the genes identified by a
650 mM, and 700 mM KCl (Fig+ 1A)+ Figure 1B shows databasesearch(Wilm&Mann,1996;Neubaueretal+,
theRNAprofileoftheelutedfractionsstainedwithethid- 1997;Shevchenkoetal+,1996;Gottschalketal+,1998)+
iumbromide;thethreeRNPpeakscorrespondtofrac- This revealed that the 25-, 27-, and 64-kD proteins of
tions 11, 20, and 24/25+The RNAs in fractions 20 and both the snR30 and snR42 complexes are Nhp2p,
24/25 are snR30 and U2, respectively+ Northern blot Gar1p,andCbf5p,respectively+Asanexamplethedata
analysisoftheseRNAfractionsconfirmedthatthe350-nt used to identify the 25-kD snR30 snoRNP protein as
RNA present in fraction 11 is snR42 (Balakin et al+, Nhp2pisshowninFigure2+Gar1pwasexpectedtobe
1996)+ThesnR42snRNPcomplexelutesinthreepeaks+ present in these complexes because it is tightly asso-
This could be because of either differences in confor- ciated with all H/ACA snoRNAs (Balakin et al+, 1996;
mation or composition of the RNPcomplex+The other Ganotetal+,1997b);however,thisisthefirsttimethat
twoabundantRNAsinfractions14,15,and16arethe Gar1p, Nhp2p, and Cbf5p have been demonstrated to
U5 snRNAs (Fig+ 1C)+ be present in biochemically purified snoRNP com-
Analysis of the protein composition of the Mono-Q plexes+
fractions on a high-TEMED 13% SDS polyacrylamide
gel revealed three major peaks (Fig+ 1D)+ The protein
Nhp2p and Cbf5p are associated
peaks in fractions 11, 20, and 24/25 cofractionate ex-
with all H/ACA-motif snoRNPs
actly with the snR42, snR30, and U2 RNAs, respec-
tively+AnalysisofthesnR30snoRNPcomplex(fraction Next we determined whether Nhp2p and Cbf5p are
20) revealed that at least four proteins with apparent associated with all H/ACA snoRNAs+ Anti-Cbf5p anti-
molecularweightsof10,25,27,and64kDarepresent bodies have been previously described that could be
in this peak+ Low amounts of Sm proteins present in usedforimmunoprecipitationstudies(Jiangetal+,1993)+
the snR30 peak are probably associated with contam- BecauseantibodiesagainstNhp2pwerenotavailable,
inating U1 snRNP complexes that also elute from the ayeaststrainwasgeneratedwherethegenomiccopy
Mono-Q column at this salt concentration (Fig+ 1D; of the gene was deleted and replaced with a plasmid
Lübbenetal+,1995)+ThesnR42snRNPcomplex(frac- expressing a hemagglutinin- (HA) tagged version of
tion11)containsfourproteinsof25,27,64,and80kD+ the protein+ Nhp2p has been demonstrated to be es-
Theelutionprofileofthe80-kDproteindoesnotexactly sentialforviability(Kolodrubetz&Burgum,1991),there-
matchthatofeitherthesnR42RNAorthe25-,27-,and fore,astheNhp2p-HAfusionproteinsupportedgrowth,
65-kDproteinsanditispossiblethatthisproteinisnot weassumethatthismodifiedproteinisfullyfunctional
partofthesnR42snoRNPcomplex+Interestingly,both (data not shown)+ Whole-cell yeast extracts were im-
the snR30 and snR42 snoRNPs share three proteins munoprecipitatedusingeithernonimmuneserum,anti-
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1552 N.J.Watkinsetal.
FIGURE1. PurificationofsnR42andsnR30
snoRNPsfromU1snRNPdepletedextracts+
Approximately300mgofanti-m3Gaffinitypu-
rifiedyeastsnRNPsandsnoRNPsfromstrain
BSY283,whichhadbeenpreviouslydepleted
ofU1snRNPs,werepassedoveraMono-Q
anion-exchangecolumn+ThesnoRNPsand
snRNPswereelutedinathree-stepgradient
from50mMto1MKClin100mLfractions+
A: Elution profile of Mono-Q purified RNP
complexes+ The KCl concentration used in
the gradient is indicated on the left of the
graphandtheabsorbanceontheright+The
fractions taken are indicated at the bottom+
The260-and280-nmabsorbancereadings
are represented by thick and thin lines, re-
spectively+TheKClgradientisindicatedbya
dashedline+B:Ethidiumbromidestainanaly-
sis of the RNA fractions+ Mono-Q fractions
were extracted with phenol/chloroform and
theRNAethanolprecipitatedfromtheaque-
ousphase+RNAswereseparatedona8%
polyacrylamide/7Mureagelandvisualized
byethidiumbromidestaining+Thepositionof
U2, snR30, snR42, and U5 RNAs are indi-
cated+Thefractionnumberisindicatedabove
eachrespectivelane+C:Northernblotanaly-
sisoftheRNAfractions+RNAsderivedfrom
each fraction were separated on a 8%
polyacrylamide/7Mureagelandtransferred
toanylonmembrane+Theblotwashybrid-
izedwithprobesagainstsnR42andU5+The
position of snR42, and U5 RNAs are indi-
cated+Thefractionnumberisindicatedabove
eachrespectivelane+D:Silverstainanalysis
oftheproteinfractions+Proteinsfromtheor-
ganicphaseofthephenol/chloroformextrac-
tion described in B were precipitated with
acetone and separated on a high-TEMED
13%polyacrylamideSDSgelandthenvisu-
alized by silver staining+ The snR42 and
snR30 snoRNP peaks correspond to frac-
tions 11 and 20, respectively+ The fraction
number is indicated above each respective
lane+LaneM:proteinmolecularmassmark-
ers the size of which are indicated at the
side+ArrowsindicatethesnR30andsnR42
snoRNP proteins+ The Sm core proteins of
U2 and other spliceosomal snRNPs are in-
dicatedbyablacksquares+
Downloaded from www.rnajournal.org on February 14, 2006
CommonH/ACAmotifsnoRNPproteins 1553
FIGURE2. Identificationofthe25-kDproteinofsnR30asNhp2pbymassspectrometry+Product-ionspectrumofthetriply
charged peptide ion at m/z 500+1+ After in-gel digestion, the resulting peptide mixture was analyzed by tandem mass
spectrometry+ Two peptides from Nhp2 were fragmented and their sequences partially determined+ Shown here is the
fragmentspectrumofthepeptide(RPTSVVFIVPGSNK),whichwasusedfordatabasesearching+FromtheB-ion
seriesB5–B9,asequencetagwasconstructed((541+4)VF(I/L)V(999+8))and,togetherwiththeintactmassofthepeptide
(1,498+2Da),wasusedtoidentifytheproteinasNhp2(SwissProtP32495)inanonredundantsequencedatabase(Mann
&Wilm,1994)+Notethattheisobaricaminoacidsisoleucineandleucinecannotbedistinguished+
Cbf5p,anti-m G,oranti-HAantibodies+TheRNAspre- are clearly coprecipitated with both Cbf5p and Nhp2p
3
cipitated by each antibody were resolved on an 8% (Fig+ 3A, lanes 4–9)+ We note that the relative amount
polyacrylamide/7Mureagelandtransferredtoanylon ofeachsnoRNAprecipitatedwitheitherCbf5porNhp2p
membrane+ The membrane was then hybridized with appears to change relative to the salt concentration
probesdirectedagainstspecificyeastRNAs(Fig+3A)+ used+ The precipitation of snoRNAs by the anti-Cbf5p
The immunoprecipitation of the HA-tagged Nhp2p antibodies appears to be more salt sensitive than with
strainwithnonimmuneserafailedtoprecipitateanyof theHAtaggedNhp2p+Webelievethatthisisbecause
the tested RNAs (Fig+ 3A, lane 2)+Also, no signal was of the reduced affinity of the anti-Cbf5p antibody for
observed when the anti-HA antibodies were used to Cbf5p at increasing salt concentrations+The reduction
precipitate an extract prepared from a yeast strain ex- in precipitation levels of snoRNAs is probably not due
pressing the wild-type (nontagged) Nhp2p (Fig+ 3A, to the destabilization of the snoRNPs at high salt con-
lane 10)+ The anti-m G (CAP) antibodies clearly pre- centrations because these H/ACA complexes are ex-
3
cipitateallcappedRNAs(Fig+3A,lane3;snR30,snR37, tremely stable and remain intact on cesium sulphate
snR42, snR11, snR10, snR3, and U3)+ However, the gradients (Lübben et al+, 1995; data not shown)+ As
anti-m Gprecipitationswereperformedat100mMKCl seen previously, a minimal amount of the U3 snoRNA
3
and the amount of U4 precipitated under these condi- is coprecipitated at low salt concentrations; however,
tions is relatively low and only seen on longer expo- theselevelsareconsiderablylowerthanpreviouslyre-
sures(datanotshown)+U14andsnR43donotcontain ported and in our hands only just visible (Fig+ 3A,
am Gcapstructureandarethereforenotprecipitated lanes4and5,anddatanotshown;Ganotetal+,1997b)+
3
bythisantibody(Fig+3A,lane3)+TheH/ACAsnoRNAs ThelowlevelcoprecipitationofU3snoRNPswasonly
snR30,snR37,snR42,snR11,snR10,snR43,andsnR3 seen at 100 mM KCl (Fig+ 3A, lanes 4 and 5); at
Downloaded from www.rnajournal.org on February 14, 2006
1554 N.J.Watkinsetal.
FIGURE3. (Legendonfacingpage.)
Downloaded from www.rnajournal.org on February 14, 2006
CommonH/ACAmotifsnoRNPproteins 1555
250 mM (Fig+ 3A, lanes 6 and 7) and 500 mM KCl andsnR13,aswellasseveralotherm Gcappedtran-
3
(Fig+3A,lanes8and9)onlytheH/ACAsnoRNPswere scripts (Fig+ 3B, lane 2)+ It is clear from this result that
precipitated+ Significantly, the box C/D U14 snoRNA an identical subset of RNAs is associated with Cbf5p,
andthespliceosomalU4snRNParenotprecipitatedat Nhp2p, and Gar1p (Fig+ 3B, lanes 3, 4, and 5)+ The
any of the tested salt concentrations (Fig+ 3A)+ This threeproteinsareassociatedwithsnR30,snR37,snR11,
datathereforesuggeststhatCbf5pandNhp2parespe- snR10, and snR31, as well as several RNAs between
cifically associated with the H/ACAclass of snoRNAs+ 182 and 211 nt in length+ The anti-fibrillarin antibodies
Becausethereareapproximately20H/ACAsnoRNAs clearly precipitate a completely different subset of
in yeast, some of which have yet to be fully charac- cellularRNAsfromthoseprecipitatedwithGar1p,Cbf5p,
terized, a slightly different approach was required to and Nhp2p (Fig+ 3B, lane 6) which include snR190,
demonstratethatthethreeproteinsNhp2p,Gar1p,and snR45, snR13 and the small antisense box C/D
Cbf5pareassociatedwithallmembersofthisclassof snoRNAs (Balakin et al+, 1996; Ganot et al+, 1997b)+
snoRNA+Therefore,theimmunoprecipitatedRNAswere From this we conclude that Gar1p, Cbf5p, and Nhp2p
39-endradiolabeledwith[32P]pCptoidentifytheRNAs are specifically associated with all of the H/ACA
associated with Cbf5p and Nhp2p+ In addition to the snoRNPs+InadditiontotheknownCguidesnoRNAs,
antibodiesusedinFigure3A,cellextractsexpressinga there appear to be several RNAs specifically associ-
proteinA-taggedGar1pwereimmunoprecipitatedwith ated with the three common H/ACAsnoRNP proteins
IgG agarose (Ganot et al+, 1997b)+ As a comparison, that are either new, uncharacterized RNAs or possibly
cell extracts were also immunoprecipitated with anti- specific degradation products+
fibrillarin antibodies (Reimer et al+, 1987)+ The radio-
labeledRNAswereseparatedona8%polyacrylamide
Genetic depletion of Nhp2p results
sequencinggel(Fig+3B)+Allimmunoprecipitationswere
in a reduction in the levels of
performed at 250 mM KCl, which appears to be the
all H/ACAsnoRNAs
minimum salt concentration required to provide speci-
ficitywithoutdramaticallyreducingthesignalseenwith Because Nhp2p is an integral snoRNP protein of the
Cbf5p(Fig+3A)+TheHA-antibodiesandtheIgGagarose H/ACA snoRNPs we decided to analyze the effect of
onlyprecipitatedaverylowamountofbackgroundtRNA depleting this protein on snoRNP biogenesis and sta-
andrRNAfromacontrolstrainthatexpressesboththe bility+ To achieve this we constructed a conditional le-
wild-type Nhp2p and Gar1p (Fig+ 3B, lanes 7 and 8)+ thal allele of NHP2, cloned under the control of the
The nonimmune serum also only precipitated a mini- GAL1promoter (Johnston & Davis, 1984)+ Growth of
malamountoftRNAfromtheHA-taggedNhp2pstrain this strain is galactose dependent and the cells do not
(Fig+ 3B, lane 1)+ The anti-m G cap antibodies specif- grow on glucose (Fig+ 4A and data not shown)+ When
3
icallyprecipitatesnR30,U1,snR42,U3,snR11,snR10, the growth rate of this conditional strain in glucose
FIGURE3. Cbf5p and Nhp2p are associated with all H/ACAsnoRNAs+ A: Whole-cell yeast extract containing either a
HA-taggedNhp2porthewild-typeproteinwereimmunoprecipitatedwithantibodiesraisedagainsttheHAepitope,Cbf5p,
them3Gcap(monoclonalantibodyH20),andcontrolnonimmuneserum+Immunoprecipitationswereperformedateither
100,250,or500mMKCl+RNAsrecoveredfromeachimmunoprecipitationwereseparatedonan8%polyacrylamide/7M
ureagelandtransferredtoanylonmembrane+TheblotwashybridizedwithprobesagainstseveralsnRNAsandsnoRNAs+
The antibody used for each immunoprecipitation is indicated above the respective lane+ NIS: nonimmune serum; CAP:
anti-m3Gantibodies(H20);Cbf5:anti-Cbf5antibodies;HA:anti-hemagglutininepitope-tagantibodies;TOT:totalRNA+Salt
concentrations used during each immunoprecipitation are also indicated+ The specific snRNA/snoRNA probe used is
indicatedtotherightofeachindividualpanel+SeveralRNAsappeartomigratefasterinthetotalRNAlanesthaninthe
precipitated lanes+ This effect is probably because of the excess of additional RNApresent in the total RNAlanes+ B:
Whole-cell yeast extract was immunoprecipitated as described above+ The HA-tagged Nhp2p cell extract was also im-
munoprecipitated with anti-fibrillarin antibodies+ In addition, the cell extracts containing a protein-A-tagged Gar1p were
immunoprecipitatedwithIgGsepharose+TheimmunoprecipitatedRNAsweresubsequentlyradiolabeledwith59-[32P]pCp
usingT4RNAligase+RadiolabeledRNAswereseparatedonan8%polyacrylamide/7Mureasequencinggelandvisualized
byautoradiography+Theantibodyusedforeachimmunoprecipitationisindicatedabovetherespectivelane+NIS:nonim-
mune sera; CAP: anti-m3G antibodies (H20); Gar1 (PA): IgG precipitation of proteinAtagged Gar1p; Cbf5: anti-Cbf5p
antibodies; Nhp2 (HA): anti-hemagglutinin epitope-tag antibody precipitation of HA-tagged Nhp2p; Fibr+: anti-fibrillarin
antibodies;PA-cont+:IgGprecipitationofwild-typeyeastextract;HA-cont+:anti-hemagglutininepitope-tagantibodyprecip-
itationofwild-typeyeastextract;Total:totalRNAfromtheHA-taggedNhp2pstrain+Itshouldbenotedthattheradiolabeled
totalRNAsfromeachyeaststrainusedintheseexperimentsgaveanidenticalpatternoflabeledRNAs(datanotshown)+
ThepositionsofthesnoRNAs,snRNAs,ribosomalRNAsandtRNAsareindicatedinaccordancewithBalakinetal+(1996)
andGanotetal+(1997b)+ThetermCguideRNAsisusedtodenotethecomplexpatternofH/ACAsnoRNAsbetween
215 and 180 nt in length and include snR43, snR44, snR35, snR34, snR5, snR46, snR3, snR189, snR8, snR32, snR9,
snR33,andsnR36+“AntisensesnoRNAs”isusedtodenotethesmallboxC/D29-O-methylaseguideRNAsthatincludeU18
andU24+
Downloaded from www.rnajournal.org on February 14, 2006
1556 N.J.Watkinsetal.
media is compared to that of the wild-type strain the
doubling times are almost identical for the first 12 h+
However, after 12 h there is a sharp decrease in the
growthoftheGAL1::NHP2strainincomparisontothe
wild-type strain (Fig+ 4A)+
To analyze the effect of depleting Nhp2p levels on
snoRNAlevels,totalRNAwasextractedfromthewild-
type and the GAL1::NHP2strain at 12-, 24-, 36-, and
48-h growth in glucose containing media+ The relative
levelsofsnoRNAsandsnRNAswerethendetermined
by Northern blot analysis (Fig+ 4B)+ Strikingly, the de-
pletion of Nhp2p results in an almost complete loss of
all H/ACA snoRNAs tested (snR30, snR37, snR42,
snR11,snR10,snR43,andsnR3)by36h+Interestingly,
thelevelsofseveralH/ACAsnoRNAs,notablysnR30,
snR3,snR10,andsnR37,appeartobesomewhatless
sensitive to the depletion of Nhp2p than others; this
mayeitherreflecttherelativeturnoverratesofindivid-
ual snoRNAs or the relative requirement of Nhp2p for
snoRNA stability+ As expected, the levels of box C/D
snoRNAs(U3andU14)andspliceosomalsnRNAs(U4)
remain unchanged+ In comparison, the depletion of
Cbf5p, another core H/ACA snoRNP protein, also re-
sults in the loss of this class of snoRNA (Lafontaine
etal+,1998),whilethedepletionofGar1phasnoeffect
on snoRNAstability (Girard et al+, 1992)+
Identification and alignment of Gar1p,
Nhp2p, and Cbf5p homologues
Gar1p, Cbf5p, and Nhp2p have been demonstrated to
be essential for viability in yeast (Kolodrubetz & Bur-
gum, 1991; Girard et al+, 1992; Jiang et al+, 1993)+As
Gar1p, Nhp2p, and Cbf5p are present in all H/ACA-
motif snoRNPs in yeast, it was expected that highly
conservedhomologuesofthesethreeproteinsexisted
inothereukaryotes+Acomparisonofthesequencesof
eachcommonproteinfromadiverserangeofeukary-
FIGURE4. Genetic depletion of Nhp2p results in a reduction of
otes should enable the primary identification of con-
H/ACAsnoRNAlevels+A:ArrestofcellgrowthbydepletionofNhp2p+
The galactose inducible strain, YNW3 (GAL::NHP2), as well as a served domains in each polypeptide+ We therefore
strain expressing Nhp2p under its own natural promoter, YNW2 screened the standard sequence databases and the
(NHP2),weregrowntomid-logarithmicphasein2%galactoseme-
expressed sequence tag (EST) database for homo-
dium+ These initial cultures were then used to inoculate glucose-
containing medium to an initial OD600 of 0+05+ The cultures were logues of these three core snoRNP proteins+
diluted accordingly to keep all OD readings below 0+5, thus main- Published Gar1p sequences from S.cerevisaeand
mtaoinninitgortehdeecveellsryin1l2oghaarinthdmpiclogttreodwaths+aThfuenOctDio6n00ooffteimaceh+Bcu:lRtuerequwiraes- Schizosaccharomycespombe(Girardetal+,1992,1993)
ment for Nhp2p in snoRNAmetabolism+ Total RNAwas extracted are aligned in Figure 5+ Included in this alignment are
fromtheyeastcellsatvarioustimesfollowingtheswitchtoglucose the new Gar1p homologues derived from a variety of
medium+RNAsrecoveredfromeachtime-pointwereseparatedon
an8%polyacrylamide/7Mureagelandtransferredtoanylonmem- highereukaryotes+Thesepolypeptidescontainseveral
brane+TheblotwashybridizedwithprobesagainstseveralsnRNAs RGGmotifrepeatsinboththeGARdomains;however,
and snoRNAs+ The yeast strains used along with the respective the number of repeats and the length of the domain
incubation time in glucose medium are indicated at the top of the
figure+ThespecificsnRNA/snoRNAprobeusedisindicatedtothe vary from protein to protein (Fig+ 5)+ Interestingly, the
rightofeachindividualpanel+Repeatexperimentsdemonstratethat central region of Gar1p is the most highly conserved
thevariationsseeninthelevelsoftotalRNAs(inparticularthe24-h part of the protein, consistent with the fact that this
time-pointfromtheNHP2strain)isbecauseofexperimentalvaria-
regionofGar1pisessentialforviabilityinyeast(Girard
tionintheamountofRNArecoveredfromindividualbatchesofyeast
cells+ etal+,1994)+ThefunctionofthiscentralregionofGar1p
is at present unclear because this region of the poly-
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CommonH/ACAmotifsnoRNPproteins 1557
FIGURE5. HomologuesofGar1pshareacentralhighlyconserveddomainbutpossessGARdomainsofvariablelength+
AminoacidalignmentoftheS.cerevisiae(accessionno+X63617),S.pombe(accessionno+Z19576),human(accession
no+AA308727),Drosophila(accessionno+S49193),Cytosporidium(accessionno+AA532317),C.elegans(accessionno+
C47427),andArabidopsis(accessionno+N65654andT21476)Gar1phomologuesusingtheClustalVprogram+Identical
residues are indicated in white on black boxes+ Conserved residues are indicated by grey boxes and are grouped as
describedinSchulz&Schirmer(1979)+ConservedRGGtripeptidesareindicatedinwhiteondarkgreyboxes+Aminoacid
positionsareindicatedontheleft+Dashedlinesindicatespacesinsertedtoaidthealignmentoftheaminoacidsequences+
Notethatthehuman,Cytosporidium,andC.eleganssequencesareincompleteattheNterminusandtheCytosporidium,
C.elegans,andArabidopsissequencesappeartobeincompleteattheCterminus+
peptide appears to have little significant homology to lishedNhp2psequenceisfromS.cerevisiae(Kolodru-
known protein-functional motifs that would predict ei- betz&Burgum,1991);additionalhomologuesfromhu-
ther RNA or protein-binding activities+ However, this man,Caenorhabditiselegans,andricewerediscovered
centraldomainprobablyissufficientfortheassociation in the database and compared by alignment (Fig+ 6)+
of this protein with the H/ACAsnoRNPs (Girard et al+, Thehumansequenceis38%identicaland56%similar
1994)+ to Nhp2p while NHP2L1 (Fig+ 6), a sequence previ-
Nhp2p was initially characterized as a high-mobility- ouslyproposedtobethehumanhomologueofNhp2p,
group-likeprotein;however,itcontainsaputativeRNA- is 27% identical and 41% similar to the yeast protein
binding motif that is also found in ribosomal proteins (Saito et al+, 1996)+ Interestingly, the NHP2L1 protein
and omnipotent suppressors of translation termination hasrecentlybeenshowntobeanessentialcomponent
(Kolodrubetz & Burgum, 1991; Koonin et al+, 1994)+ ofthespliceosome(S+Nottrott,P+Fabrizio,andR+Lühr-
Thisproteinisproposedtohaveamassof17+1kDbut mann, unpubl+)+ We therefore propose that the new
migrates at 25 kD on a high-TEMED SDS polyacryl- humansequence,andnotNHP2L1,isthehumanortho-
amide gel+ Interestingly, in vitro translated Nhp2p also logue of Nhp2p+
migratesat25kDonahigh-TEMEDSDSgel(datanot The sequence homology seen between the Nhp2
shown);however,itisnotunusualforahighlycharged protein candidates from several eukaryotic species is
protein(Nhp2phasanestimatedpIof9+73)tomigrate extremelyhigh+IthasbeenobservedthatseveralRNP
anomalously on polyacrylamide gels+ The only pub- proteins, including Nhp2p, share a conserved region/