Trans-regulation of the expression of the transcription factor MtHAP2-1 by a uORF controls root ...

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Trans-regulation of the expression
of the transcription factor MtHAP2-1
by a uORF controls root nodule
development
JeanPhilippeCombier,1,3 FrançoisedeBilly, PascalGamas, AndreasNiebel, and SusanaRivas2
LaboratoiredesInteractionsPlantesMicro-organismes(LIPM),CentreNationaldelaRechercheScientifique-Institut
NationaldelaRechercheAgronomique(CNRS-INRA)UMR2594/441,F-31320CastanetTolosan,France
MtHAP2-1isaCCAAT-bindingtranscriptionfactorfromthemodellegumeMedicagotruncatula.We
previouslyshowedthatMtHAP2-1expressionisregulatedbothspatiallyandtemporallybymicroRNA169.
HerewepresentanovelregulatorymechanismcontrollingMtHAP2-1expression.Alternativesplicingofan
intronintheMtHAP2-15(cid:1)leadersequence(LS)becomespredominantduringthedevelopmentofrootnodules,
leadingtotheproductionofasmallpeptide,uORF1p.OurresultsindicatethatbindingofuORF1pto
MtHAP2-15(cid:1)LSmRNAleadstoreducedaccumulationoftheMtHAP2-1transcriptandmaycontributeto
spatialrestrictionofMtHAP2-1expressionwithinthenodule.WeproposethatmiR169anduORF1pplay
essential,sequential,andnonredundantrolesinregulatingMtHAP2-1expression.Importantly,incontrastto
previouslydescribedcis-actinguORFs,uORF1pisabletoactintranstodown-regulategeneexpression.Our
workthuscontributestoabetterunderstandingoftheactionofupstreamORFs(uORFs)intheregulationof
geneexpression.
[Keywords:HAP2;Medicagotruncatula;alternativesplicing;symbiosis;transcriptionfactor;upstreamORF]
Supplementalmaterialisavailableathttp://www.genesdev.org.
ReceivedOctober29,2007;revisedversionacceptedApril8,2008.
Alternativesplicing(AS)isanimportantpost-transcrip- todes,forexample).Indeed,thehumangenomecontains
tional regulatory mechanism contributing to the func- significantly fewer genes than was initially predicted at
tionalcomplexityofhighereukaryotes.Itcanparticipate the outset of the Human Genome Project, with only
in increasing protein diversity by generating multiple ∼1.5%ofthetotallengthservingasprotein-codingexons
transcripts from a single gene (Graveley 2001; Kazan (International Human Genome Sequencing Consortium
2003). In addition, AS can reduce gene expression by 2001). However, human genes are more complex, with
yielding isoforms that are degraded by nonsense-medi- AS generating a larger number of protein products. In
ated mRNA decay (NMD) or other mechanisms (Hill- certaincases,onegenecangeneratethousandsofspliced
manetal.2004).Insomecases,ASoccurswithinthe5(cid:2) variants (Soller 2006). Traditional gene-by-gene investi-
leader sequence (LS) of the gene, leading to post-tran- gationsofASmechanismsarenowbeingcomplemented
scriptionalregulationorinhibitionoftranslation,aswell byglobalapproaches(Matlinetal.2005;Blencowe2006;
astotheproductionofproteinswithdifferentproperties Cuperlovic-Culf et al. 2006) and the publication of AS
(Procissi et al. 2002; Halterman et al. 2003; Hu et al. databases (http://www.ebi.ac.uk/asd). Recent genome-
2005;Robertsetal.2005). wide studies have indicated that up to 70% of human
Overallorganismcomplexitydoesnotnecessarilycor- genesundergoAS(Baeketal.2005).Abigproportionof
relate with the complexity of the respective genome these genes encode proteins involved in signaling and
(∼25,000 genes in humans and ∼19,000 genes in nema- generegulation(ModrekandLee2002).
Althoughourknowledgeoftheextentofthisphenom-
enoninhigherplantsisstillpoor,recentcomputational
andexperimentalstudiessuggestthatASprobablyplays
1Presentaddress:InstitutNationaldelaSantéetdelaRechercheMédi- afarmoresignificantroleinthegenerationofplantpro-
cale (INSERM) U563, Department Lipoprotéines et Médiateurs Li-
pidiques,HôpitalPurpan,31024Toulouse,France teome diversity than was previously thought. The pro-
Correspondingauthors. posal that 21% of all predicted Arabidopsis genes are
[email protected];FAX[phone].
alternativelyspliced(WangandBrendel2006)isprobably
[email protected];FAX[phone].
Articleisonlineathttp://www.genesdev.org/cgi/doi/10.1101/gad.461808. anunderestimatesince(1)thenumberofpubliclyavail-
GENES&DEVELOPMENT22:1549–1559©2008byColdSpringHarborLaboratoryPressISSN0890-9369/08;www.genesdev.org 1549
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Combieretal.
able expressed sequenced tags (ESTs) from Arabidopsis expression is regulated by a microRNA, miR169 (Com-
(∼1.5million)isfarbelowthatforotherrelativelywell- bieretal.2006).Thecomplementaryexpressionpattern
studied genomes (nearly 8 million ESTs for the human ofMtHAP2-1andmiR169indicatesthatthefunctionof
genome; http://www.ncbi.nlm.nih.gov/dbEST/dbEST_ miR169maybetorestrictMtHAP2-1expressiontothe
summary.html), (2) full-length cDNAs are available for meristematic zone as part of a process that is essential
onlyapproximatelyhalfofallpredictedgenes(Chunget fornoduledifferentiation(Combieretal.2006).
al. 2006), and (3) ESTs from genes with low expression Here we describe a previously unknown post-tran-
levels or with expression at a particular developmental scriptionalmechanismthatregulatesMtHAP2-1expres-
stage or physiological condition, which are more likely sionduringnodulation.ASofanintronintheMtHAP2-1
tobealternativelyspliced,arelikelytobepoorlyrepre- 5(cid:2)LS increases during nodule development, leading to
sentedinthedatabases. theproductionofasmallpolypeptide,uORF1p.Interest-
Among the various cis elements in mRNAs that par- ingly, in contrast to previously described cis-acting
ticipateinregulatingtranslationareAUGcodonswithin uORFs,uORF1pisabletoactintranstodown-regulate
transcript leaders (upstream AUGs) and, in some cases, gene expression. We present a model in which both
associated upstream ORFs (uORFs). Up to 50% of eu- miR169anduORF1pcontributetospatialandtemporal
karyoticmRNAscontainAUGcodonswithintheirtran- regulationofMtHAP2-1expressionduringnoduledevel-
scriptleaderregions(Suzukietal.2000).uAUGsarecon- opment.
spicuously common in genes involved in the control of
cellulargrowthanddifferentiation,includingoncogenes
andtranscriptionfactorgenes(Kozak1987,1991,2002).
Results
The best documented mode of action of uORFs is the
inhibitionoftranslationthroughamechanismcalledri-
ASoftheMtHAP2-1intron1,locatedinthe5(cid:2)LS
bosomestalling.Inthiscase,theribosomestallsduring
either the elongation or termination phase of uORF The 5(cid:2)LS of MtHAP2-1 comprises two short exon se-
translation, creating a block in subsequent ribosome quences,E1(33basepairs[bp])andE2(55bp),flankinga
scanning. large intron, I1 (865 bp) (Fig. 1A). Analysis of the avail-
The symbiotic interaction between leguminous plant ableMedicagotruncatulaESTdatabases(http://medicago.
speciesandbacteriacollectivelyknownasrhizobialeads toulouse.inra.fr/Mt/EST) identified a set of eight ESTs
to the de novo development of differentiated organs, corresponding to MtHAP2-1. Interestingly, one of these
known as nodules, on the roots of host plants. A fine- (MtBB39H03) contains part of the MtHAP2-1 5(cid:2)leader
tuned molecular dialogue is established between plant intron sequence, suggesting that AS may occur in the
andbacteriaduringnodulation,forwhichkeyplayersare MtHAP2-15(cid:2)LS.
the bacterial-produced lipooligosaccharides called Nod Real-time RT–PCR analysis showed that MtHAP2-1
factors.Fourmaindifferentiatedzonescanbeidentified expression is very low in noninoculated M. truncatula
inamaturenodule:themeristematicapicalzone(aplant roots. During nodule development, MtHAP2-1 expres-
stemcellcompartment),theinfectionzone,thefixation sioninnodulespeaks4dafterinoculationwithSinorhi-
zone,andthesenescentzone(Vasseetal.1990;Patriarca zobium meliloti, and then expression decreases during
etal.2004).Althoughearlynodulationevents,including laterstagesofnodulation(Fig.1B;Combieretal.2006).
Nod factor signaling and infection, have been exten- SinceASofintronslocatedin5(cid:2)LSsmayhaveanimpor-
sively investigated, later stages of nodulation such as tant effect on gene expression and regulation (Lahousse
meristem differentiation and nodule organogenesis re- etal.2004;Robertsetal.2005),wefurtherinvestigated
main poorly understood (Foucher and Kondorosi 2000; AS of the intron I1 in the MtHAP2-1 5(cid:2)LS. Primers
Patriarcaetal.2004). within either the intron1 or the exon1–exon2 junction
We previously identified MtHAP2-1, a HAP2-type wereusedtoquantifytheexpressionofthealternatively
transcription factor of the CCAAT-box-binding family and normally spliced forms of MtHAP2-1, respectively.
(CBF), called NF-Y in humans and HAP in plants and Asacontrol,PCRusingprimershybridizingwithinthe
yeast (El Yahyaoui et al. 2004). HAP proteins form het- fourthintron(I4)ofMtHAP2-1resultedinnoamplifica-
erotrimericcomplexesthatbindtotheCCAATconsen- tion,allowingustoruleoutthepresenceofcontaminat-
susmotifthatispresentin30%ofeukaryoticpromoters ing genomic DNA. As shown in Figure 1C, low expres-
(Mantovani1998).HAPproteincomplexesappeartoplay sion of MtHAP2-1 in roots correlates with the presence
centralrolesduringdevelopmentbyregulatingcelldivi- ofthealternativelysplicedforminnoninoculatedroots
sion and/or differentiation in eukaryotes (Bhattacharya andinnodules14and21dpi,whereasinyoungnodules
etal.2003;DiAgostinoetal.2006).Inplants,HAPgenes (4 dpi), which show the highest level of MtHAP2-1 ex-
havebeenshowntobekeyregulatorsofembryogenesis, pression, about 90% of MtHAP2-1 is normally spliced.
chloroplast biogenesis, flowering-time control, or blue Intriguingly, AS increases during nodule development,
light and abscisic acid responses, for example (Lotan et withthealternativelysplicedformreachingabout50%
al. 1998; Kwong et al. 2003; Lee et al. 2003; Miyoshi oftotalMtHAP2-1expression21dafterinoculation.Fur-
et al. 2003; Ben-Naim et al. 2006; Warpeha et al. 2007). thermore,Westernblotanalysisusingapolyclonalanti-
MtHAP2-1playsakeyroleduringnoduledevelopment, MtHAP2-1antibodyrevealedthat,asfortheMtHAP2-1
possiblybyregulatingnodulemeristemfunction,andits transcript, MtHAP2-1 protein expression in nodules
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MtHAP2-1intrans-regulationbyauORF
Figure1. MtHAP2-1intron1isalternativelyspliced.(A)SchematicrepresentationofMtHAP2-1genestructure.Exons(E1–E6)are
representedwithboxesandintrons(I1–I5)areshownasthicklines.MtHAP2-1firstintron(I1),shownwithadiscontinuousbar,is
locatedinthe5(cid:2)LS.Sequenceofthe5(cid:2)LSisshownatthebottomwithexonsE1andE2underlined.Deducedaminoacidsequences
ofuORF1(red),uORF2(blue),anduORF3(gray),areshownunderthenucleotidesequence.(B)ExpressionanalysisofMtHAP2-1during
nodule development. MtHAP2-1 transcript levels were determined by qRT–PCR with cDNA obtained from M. truncatula roots
inoculatedwithS.melilotiattheindicatedtimepoints.TheexpressionvalueswerenormalizedusingtheexpressionlevelofMtEF1(cid:1)
asaninternalstandard(ElYahyaouietal.2004)andrelatedtothevalueofMtHAP2-1expressionlevelbeforeinoculation(0dpi),which
issetat1.MeanexpressionvaluesandSEMvalueswerecalculatedfromtheresultsofthreeindependentexperiments.(C)qRT–PCR
analysisoftheexpressionlevelsofalternatively(darkgray)andnormallyspliced(lightgray)MtHAP2-1transcripts.Theexpression
valueswerenormalizedusingtheexpressionlevelofMtEF1(cid:1)asaninternalstandardandrelatedtothevalueofthetotalMtHAP2-1
geneexpressionateachtimepoint,whichissetat100%.(D)MtHAP2-1proteinexpressionanalysis.(Toppanel)Totalproteinsfrom
noduleswereextractedattheindicatedtimepoints,expressedindaysafterinoculation,andproteinextracts(50µg)wereseparated
and analyzed by immunoblotting using anti-MtHAP2-1 polyclonal antibodies. (Bottom panel) Equal loading in each lane was con-
firmedbyPonceauSstaining.
peaks 4 d after inoculation and then decreases during gene, allowing segregation between the unspliced and
nodule development (Fig. 1D). Interestingly, this de- spliced variants (Celotto and Graveley 2002). To test
crease in MtHAP2-1 protein levels correlates with an the functional role of AS of MtHAP2-1 during nodule
increaseoftheMtHAP2-1alternativelysplicedform,14 development, an RNAi construct was generated using
and 21 dpi. In addition, qRT–PCR analysis showed that a sequence located in the first intron of MtHAP2-1
the weak expression of MtHAP2-1 in different plant or- (RNAiintron1). Specific silencing of MtHAP2-1-AS was
gans correlates with the presence of the alternatively confirmedbyqRT–PCRanalysis(SupplementalFig.S1).
splicedform(datanotshown).Takentogether,thesere- We showed previously that silencing of both alterna-
sults suggest that AS of the intron 1 of MtHAP2-1 is tivelyandnormallysplicedformsofMtHAP2-1,usinga
regulatedduringnoduledevelopmentandmayresultin sequence located in the 3(cid:2)UTR (RNAiMtHAP2-1), led to
reducedMtHAP2-1accumulation. theformationofsmall,nonfunctional(non-nitrogen-fix-
The alternatively spliced version of MtHAP2-1, con- ing)nodules,arrestedingrowth(Combieretal.2006).In
tainingintron1,willbehereafterreferredtoasMtHAP2- contrast, nodules formed on transgenic roots carrying
1-AS and the normally spliced form will be called the RNAiintron1 silencing cassette are nitrogen fixing,
MtHAP2-1-NS. with a normal differentiated structure and an active
and persistent meristem (Fig. 2A). Nevertheless, due to
the small size of these RNAiintron1 nodules compared
SpecificsilencingofMtHAP2-1-ASleadstoanodule
with control nodules, it is clear that growth has been
growthdefect
significantly modified. Importantly, MtHAP2-1 tran-
RNAihasbeensuccessfullyusedinculturedDrosophila script and protein levels in these nodules, 21 d after in-
cellstospecificallysilencetheunsplicedformofagiven oculation, were significantly higher than for wild-type
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Combieretal.
plants(Fig.2B,C).TheseresultsimplythattheMtHAP2- OverexpressionofMtHAP2-1withoutitsregulatory
1-AS is required for normal nodule development, and sequencesresultsinsmall,nitrogen-fixingnodules
suggest that this form negatively regulates MtHAP2-1
Our results indicate that both MtHAP2-1 5(cid:2) and 3(cid:2)-ter-
transcriptandproteinaccumulation.
minalsequencesplaykeyrolesintheregulationofnod-
ule development. To further test this idea, MtHAP2-1,
without its 5(cid:2) and 3(cid:2) regulatory regions, was overex-
pressed in M. truncatula roots. Overexpression of
MtHAP2-1 in the transgenic roots was confirmed by
both qRT–PCR and Western blot analysis using anti-
MtHAP2-1 polyclonal antibodies (Supplemental Fig.
S2B,C).Transgenicrootsdevelopednitrogen-fixingnod-
ules, with a normal and differentiated structure. How-
ever, nodule size was again significantly reduced, as
compared with the controls (Supplemental Fig. S2A).
Thisresultprovidesfurtherevidencethatalteredspatial
andtemporalexpressionofMtHAP2-1resultsinaltered
noduleorganogenesis.
AGUSfusionrevealsthatuORF1isexpressed
inthenoduleinfectionzone
AnalysisofthesequenceofthefirstintronofMtHAP2-1
revealed11uORFspotentiallyencodingpolypeptidesof
between4and62aminoacids.Wefocusedouranalysis
on the three longest ORFs, named uORF1, uORF2, and
uORF3,potentiallyencodingpolypeptidesof62,50,and
34aminoacids,respectively(Fig.1).Ifthefirstintronis
unspliced, ribosome scanning could initiate translation
attheuAUGscodonsoftheseuORFsratherthanatthe
AUG at the start of the main MtHAP2-1 ORF. To test
this hypothesis, ∼2.3-, ∼2.4-, and ∼2.6-kb upstream
MtHAP2-1genomicpromotersequencesextendingasfar
as the uATGs of uORF1, uORF2, and uORF3, respec-
tively, were fused to a GUS reporter gene and M. trun-
catula roots were transformed with these three con-
structs. Whereas roots expressing fusions with uORF2
and uORF3 did not show any GUS expression (data not
shown),thefusionwithuORF1revealedGUSexpression
(red staining) located in the infection zone of mature
nodules (zone II) (Fig. 3A) but not in the meristematic
zone(zoneI)whereMtHAP2-1isexpressed.Thisobser-
Figure 2. RNAiintron1 transgenic roots show a nodule growth vation strongly suggests that the AUG of uORF1 can
defect.(A)Lightmicroscopyimagesoflongitudinalsections(50 initiate translation, and therefore may lead to the syn-
µm) of nodules of M. truncatula transgenic roots expressing thesis of the 62-amino-acid polypeptide in nodule tis-
eitheranRNAiintron1construct(right)oranempty-vectorcon- sues.
trol (left). Pictures were taken 21 d after inoculation with S.
meliloti.Bar,100µm.(B)MtHAP2-1transcriptlevelswerede-
terminedbyqRT–PCRwithcDNAobtainedfromempty-vector OverexpressionofuORF1leadstoaseverenodule
control, and RNAiintron1 M. truncatula nodules 21 d after in-
developmentaldefect
oculationwithS.meliloti.Theexpressionvalueswerenormal-
izedusingtheexpressionlevelofMtEF1(cid:1)asaninternalstan- To obtain more functional information, uORF1 was
dardandrelatedtothevalueoftheMtHAP2-1transcriptlevelin overexpressedinM.truncatularoots,underthecontrol
theRNAiintron1transgenicplants,whichissetat100%.Mean of the constitutive 35S promoter. Very small nodules
expression values and SEM values were calculated from the
were formed on transgenic roots 21 d after inoculation,
results of two independent experiments. (C, top panel) Total
andnodulesectioningrevealedonlypoorlydifferentiated
proteins from empty-vector control and RNAiintron1 M. trun-
zones, enclosed by the endodermis, thus indicative of
catula nodules were extracted 21 d after inoculation with S.
early growth arrest (Fig. 4A). Importantly, MtHAP2-1
meliloti,andproteinextracts(50µg)wereanalyzedbyimmu-
noblotting using anti-MtHAP2-1 polyclonal antibodies. (Bot- transcript and protein levels were greatly reduced in
tompanel)EqualloadinginbothlaneswasconfirmedbyPon- thesenodules5dafterinoculation(Fig.4B,C).Thisphe-
ceauSstaining. notypeisverysimilartothatobservedinRNAiMtHAP2-1
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MtHAP2-1intrans-regulationbyauORF
clearspecificityforuORF1actionontheMtHAP2-15(cid:2)LS
(SupplementalFig.S4).ToconfirmthatuORF1isindeed
translated in N. benthamiana, an HA-tagged version of
Figure3. uORF1isexpressedintheinfectionzoneofmature
nodules. Histochemical localization of (cid:2)-glucuronidase (GUS)
activity (A, red color) and nuclear staining using DAPI (B) of
longitudinalsectionsofnodulesfromM.truncatulatransgenic
rootsexpressingauORF1-GUSfusionconstruct.Dark-field(A)
and fluorescence (B) microscopy images were taken 21 d after
inoculation with S. meliloti. (B). Differentiated zones can be
distinguishedinthematurenoduleafternuclearstainingwith
DAPI (Vasse et al. 1990). (I) Meristematic zone. (II) Infection
zone.(III)Nitrogenfixationzone.Bar,50µm.
nodules,forwhichMtHAP2-1transcriptandproteinlev-
els are also significantly reduced (Fig. 4B,C; Combier et
al.2006).ThesedataimplythatuORF1canactintrans
to negatively regulate MtHAP2-1 accumulation, and
confirmthatthepreciseregulationofMtHAP2-1expres-
sionisrequiredfornormalnoduledevelopment.
ExpressionoftheuORF1proteinintransleads
toreducedaccumulationofaMtHAP2-15(cid:2)LS-AS
containingtranscript
To gain further insight into the control of MtHAP2-1
expressionbyuORF1,a35S:GUSfusionconstruct,with
orwithoutthealternativelyspliced,intron1-containing,
MtHAP2-1 5(cid:2)LS (Fig. 5A), was coexpressed with either
35S:uORF1, 35S:uORF2, or 35S:uORF3 in Nicotiana Figure 4. Overexpression of uORF1 in roots leads to
benthamiana leaves. Both MtHAP2-1 5(cid:2)LS-AS and MtHAP2-1 degradation and a defect in nodule organogenesis.
MtHAP2-1 5(cid:2)LS-NS transcripts are detected after tran- (A)Lightmicroscopyimages(50µm)oflongitudinalsectionsof
sient delivery of a MtHAP2-1 5(cid:2)LS-AS-GUS fusion nodules of M. truncatula transgenic roots expressing either a
35S:uORF1 construct (right) or an empty-vector control (left).
(SupplementalFig.S3),confirmingthatASofMtHAP2-1
Picturesweretaken21dafterinoculationwithS.meliloti.Bar,
5(cid:2)LSoccursinN.benthamianaandvalidatingourstrat-
100 µm. (B) MtHAP2-1 transcript levels were determined by
egy to use N. benthamiana as a heterologous system. qRT–PCR with cDNA obtained from empty-vector control,
Importantly, overexpression of uORF1 led to decreased 35S:uORF1andRNAiMtHAP2-1M.truncatulatransgenicroots5
GUSexpressionandreducedGUStranscriptandprotein dafterinoculationwithS.meliloti.Theexpressionvalueswere
levels only when coexpressed with the 35S:5(cid:2)LS-GUS normalizedusingtheexpressionlevelofMtEF1(cid:1)asaninternal
construct(Fig.5B–D,lanes1,2).ThehigherGUSexpres- standard and related to the value of the MtHAP2-1 transcript
sionlevelobservedwiththe35S:5(cid:2)LS-AS-GUScompared levelinthecontrolplants,whichissetat100%.Meanexpres-
sionvaluesandSEMvalueswerecalculatedfromtheresultsof
with the 35S:GUS construct is probably due to intron-
two independent experiments. (C, top panel) Total proteins
mediatedenhancement,aphenomenonthathasbeenre-
from empty-vector control, 35S:uORF1 and RNAiMtHAP2-1
portedtoconferupto100-foldenhancementofgeneex-
M.truncatulatransgenicrootswereextracted5dafterinocu-
pression(Kimetal.2006).NoeffectonGUSexpression
lation with S. meliloti, and protein extracts (50 µg) were ana-
was detected when uORF1 was coexpressed with lyzedbyimmunoblottingusinganti-MtHAP2-1polyclonalan-
35S:GUS-3(cid:2)UTR (data not shown) or when uORF2 or tibodies. (Bottom panel) Equal loading was confirmed by Pon-
uORF3 were used for coinfiltration, thus indicating a ceauSstaining.
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Combieretal.
Taken together, these data provide strong evidence
thattheuORF1ppeptideactsintranstodown-regulate
MtHAP2-1expressionbytargetingthe5(cid:2)LS.
uORF1pbindstobothMtHAP2-1-AS
andMtHAP2-1-NS-containingmRNA
To investigate whether uORF1p can interact directly
with the MtHAP2-1 5(cid:2)LS-containing mRNA, we made
use of an RNA pull-down assay. uORF1p, transiently
expressedinN.benthamianaleaves,wasfoundtobind
bothMtHAP2-15(cid:2)LS-ASandMtHAP2-1-NSbiotinylated
RNA(Fig.6A,lanes2,3).Thespecificityofthisbinding
was demonstrated by the inability of uORF1p-HA to
bind to the streptavidin resin alone (data not shown) or
afterincubationwithabiotinylatedcontrolRNAofthe
samelengthcorrespondingtotheunrelatedArabidopsis
transcriptionfactorAtMYB30(Fig.6A,lane4).Interest-
ingly,nouORF1pbindingwasdetectedafterincubation
Figure5. uORF1pexpressioninN.benthamianaleavesleads
toreducedlevelsofGUSfusedtoMtHAP2-15(cid:2)LS-AS.Expres-
sionanalysisofGUS((cid:2)-glucuronidase)2dafterAgrobacterium-
mediatedtransientexpressionoftheGUSfusionsshowninA
together with either an empty vector control (1), untagged
35S:uORF1 (2), or 35S:uORF1-HA (3). (A) Schematic represen-
tationoftheGUSfusionsusedinthisexperiment.MtHAP2-1
5(cid:2)LS,containingthefirstintron(discontinuousline),wasfused
tothe(cid:2)-glucuronidase(GUS)geneunderthecontrolofthe35S
promoter. (B) GUS expression determined by GUS histo-
chemicalstainingofN.benthamianaleafdiscs.(C)GUStran-
script levels determined by qRT–PCR. The expression values Figure6. uORF1pbindsspecificallytoMtHAP2-15(cid:2)LSRNA.
were normalized using the expression level of NbEF1(cid:1) as an
WesternBlotswithananti-HAantibodyshowinguORF1p-HA
internal standard and related to the expression value of
recoveredaftertheRNApull-downassays.(A,lane1)Inputof
35S:5(cid:2)LS-GUS+untagged 35S:uORF1, which is set at 100%.
uORF1p-HAcorrespondsto10%ofthetotalN.benthamiana
MeanexpressionvaluesandSEMvalueswerecalculatedfrom
protein extract used for each binding assay. uORF1p-HA was
theresultsofsevenindependentexperiments.(D)Westernblot
elutedfromthestreptavidinbeadsafterincubationwithbioti-
analysis showing GUS protein (middle panel) and uORF1-HA nylatedMtHAP2-15(cid:2)LS-AS-corresponding(lane2)orMtHAP2-
(toppanel)expressionlevels.PonceauSstainingofribulose-1,5-
1-NS-corresponding(lane3)RNA.uORF1p-HAwasnoteluted
bisphosphatecarboxylase/oxygenase(rubisco)forconfirmation
fromthestreptavidinbeadsafterincubationwiththebiotinyl-
ofequalloadingineachlaneisshownatthebottom. ated AtMYB30- (lane 4) orMtHAP2-1 (cid:3)5(cid:2)LS-corresponding
RNA(lane5).(B)IncreasingamountsoftotalN.benthamiana
proteinextractsexpressinguORF1p(1mg,lane1;5mg,lane2;
uORF1 was generated. As for the untagged version of and25mg,lane3)wereusedforthebindingassay.uORF1p-HA
uORF1, expression of 35S:uORF1-HA led to decreased was eluted from the streptavidin beads after incubation with
GUSexpression,andreducedGUStranscriptandprotein biotinylated MtHAP2-1 5(cid:2)LS-AS-corresponding RNA. (C) In-
levels, only when coexpressed with the GUS construct creasing ratios of biotinylated to nonbiotinylated MtHAP2-1
containing the MtHAP2-1 5(cid:2)LS (Fig. 5B–D, lanes 1,3). 5(cid:2)LS-AS-correspondingRNA(0:1,lane1;1:3,lane2;1:1,lane3;
Expression of the uORF1-HA fusion protein was con- and1:0,lane4)wereusedforincubationwiththestreptavidin
beadspriortobindingofuORF1p-HA.(D)Competitionexperi-
firmedbyWesternblottingusinganti-HAantibodies.No
effect on the expression of 35S:5(cid:2)LS-GUS was observed ment in which uORF1p was first bound to streptavidin beads
containing a 1:1 mix of biotinylated to nonbiotinylated
whenusingamutatedformofuORF1,inwhichtheATG
MtHAP2-1 5(cid:2)LS-AS RNA (input of uORF1p, lane 1) prior to
startcodonhadbeenmutatedtoAGGtopreventprotein elution using MtHAP2-1 5(cid:2)LS-AS (eluted uORF1p, lane 2) or
translationinitiation(SupplementalFig.S4).Finally,we AtMYB30nonbiotinylatedRNAs(eluteduORF1p,lane4).The
found that uORF1p is able to down-regulate expression amountofuORF1premainingonthebeadsaftertreatmentwith
ofbothMtHAP2-15(cid:2)LS-ASandMtHAP2-15(cid:2)LS-NS-con- MtHAP2-15(cid:2)LS-AS(lane3)orAtMYB30 nonbiotinylatedRNAs
tainingtranscripts(SupplementalFig.S3). (lane5)isalsoshown.
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MtHAP2-1intrans-regulationbyauORF
with a biotinylated RNA corresponding to MtHAP2-1 the RNAiMtHAP2-1 silencing cassette or overexpres-
deletedfromits5(cid:2)LS(MtHAP2-1(cid:3)5(cid:2)LS)(Fig.6A,lane5), sing miR169 or uORF1p possess significantly reduced
strongly suggesting that uORF1p physically interacts MtHAP2-1 levels that correlate with precocious nodule
with the E1 and/or E2 exon sequence(s) present in meristem abortion, whereas roots expressing the
MtHAP2-15(cid:2)LS(Fig.1). RNAiintron1 silencing cassette or a miR169-resistant
uORF1pwasabletobindMtHAP2-15(cid:2)LS-ASbiotinyl- form of MtHAP2-1, or overexpressing MtHAP2-1 with-
ated RNA in a dose-dependent manner (Fig. 6B, lanes out its regulatory regions, possess increased MtHAP2-1
1–3).NouORF1pwaselutedfromthebeadswhenanon- levels that correlate with a nodule growth defect. Al-
biotinylated MtHAP2-1-AS RNA was used for incuba- though one could argue that nodule growth deficiency,
tionwiththestreptavidinresin(Fig.6C,lane1).Differ- in RNAiintron1 or 35S:uORF1 transgenic plants, might
ent ratios of nonbiotinylated to biotinylated MtHAP2-1 lead to altered MtHAP2-1 expression, this seems un-
5(cid:2)LS-ASRNAswereusedforbindingtothestreptavidin likely,sinceMtHAP2-1expressionisunalteredinMedi-
beads, further demonstrating the quantitative character cago mutants presenting a reduced nodule growth phe-
of uORF1p binding to MtHAP2-1 5(cid:2)LS (Fig. 6C, lanes notype (T. Vernie, J.-P. Combier, P. Gamas, and A.
1–4). Finally, a competition experiment was performed Niebel,unpubl.),suchaslin(Kuppusamyetal.2004)and
inwhichuORF1pwasfirstboundtoMtHAP2-15(cid:2)LS-AS nip (Veereshlingam et al. 2004). We therefore conclude
RNA-containing beads (Fig. 6D, lane 1) and then par- that nodule growth defect in RNAiintron1 or 35S:uORF1
tially eluted from the resin using a nonbiotinylated plantsis(1)notsufficienttoexplainthereducedexpres-
MtHAP2-15(cid:2)LS-AS-(Fig.6D,lane2)butnotanonbioti- sionofMtHAP2-1and(2)duetoreducedMtHAP2-1ex-
nylatedAtMYB30-(Fig.6D,lane4)correspondingRNA. pression,andnotviceversa.
Figure 6D also shows the amount of uORFp1 protein We propose that restriction of MtHAP2-1 expression
that remained attached to the beads after competition to the meristematic zone of the nodule is essential for
with both RNAs (nonbiotinylated MtHAP2-1 5(cid:2)LS-AS normalnoduledevelopment,andthatthisisachievedby
[lane3]andAtMYB30[lane5]). both miR169 targeting of the MtHAP2-1 3(cid:2)UTR and al-
TheseresultsclearlyshowthatuORF1pisabletobind ternativeprocessingoftheMtHAP2-15(cid:2)LS.Thepattern
inaquantitativemannertoanmRNAcontainingeither ofbothmiR169expressionandASoccurrenceinnodules
the alternatively or the normally spliced MtHAP2-1 suggests that the control of MtHAP2-1 expression is
5(cid:2)LS, and suggest that this could be the mechanism finelyregulated.miR169expressionandASappeartobe
by which uORF1p mediates the negative regulation of complementary from a temporal point of view, since
MtHAP2-1expressioninplanta. miR169 transcript abundance peaks 10 d after inocula-
tion and decreases later during nodule formation
(SupplementalFig.S5),whereasASincreasesduringnod-
Discussion
uledevelopment,reachingamaximum21dafterinocu-
Inthisstudy,weshowthatASofintron1,presentinthe lation(Fig.1C;SupplementalFig.S5).
5(cid:2)LSofMtHAP2-1,regulatesMtHAP2-1expressiondur- Figure 7 proposes a model for the regulation of
ing nodule formation. Unlike the many examples de- MtHAP2-1 expression. In this model, miR169 and
scribed for Drosophila and Caenorhabditis elegans, uORF1 play essential, sequential, and nonredundant
thereareveryfewcasesofdevelopmentalswitchesbeing roles in the control of MtHAP2-1 expression, with
controlledbyASinplants,andmoreparticularly,inle- miR169 acting during the early stages of nodule devel-
gumes (Lorkovic et al. 2000; Macknight et al. 2002; opment and uORF1p taking over this function later in
Cheungetal.2006).RegulationofMtHAP2-1expression nodulation. Our data show that uORF1p acts on both
thusprovidesagoodmodeltodeterminethemolecular alternatively and normally spliced MtHAP2-1 tran-
mechanisms that have evolved in plants to control pre- scripts,since(1)silencingofMtHAP2-1-ASalsoleadsto
mRNAprocessing. the accumulation of MtHAP2-1-NS (Supplemental Fig.
S1), (2) uORF1p is able to down-regulate the expression
of both the alternatively and normally spliced forms of
AdualregulationbymiR169anduORF1pcontrols
MtHAP2-1 5(cid:2)LS in N. benthamiana (Supplemental Fig.
MtHAP2-1expressionandnodulefunction
S3), and (3) uORF1p binds to both MtHAP2-1-AS and
Datapresentedhere,togetherwithourpreviousworkon MtHAP2-1-NS-correspondingRNAs(Fig.6A).Takento-
regulationofMtHAP2-1bymiR169,suggestthatadual gether, these results strongly suggest that E1 and/or E2
regulation by miR169 and uORF1p controls MtHAP2-1 exons are the target sequences of uORF1p binding for
expression.WepreviouslydemonstratedthatmiR169is regulationofMtHAP2-1expression.
expressedintheinfectionzoneofthenodule(Combieret
al.2006).Likewise,GUSexpressionintheinfectionzone
ExpressionofMtHAP2-1iscontrolledbyanovel
of nodules from uORF1-GUS transgenic plants (Fig. 3)
post-transcriptionalregulatorymechanism
strongly suggests that ORF1p expression also localizes
to this region of the nodule. Misregulation of either uORF-mediated regulation of gene expression is well
miR169 or uORF1p expression has a major effect on studied,especiallyinanimals,whereinhibitionoftrans-
MtHAP2-1transcriptandproteinexpression,whichcor- lationisthebestdocumentedmodeofaction(Morriset
relates with a nodule growth defect: Roots expressing al. 2000; Kozak 2002). Translation of a uORF can pro-
GENES&DEVELOPMENT 1555
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Combieretal.
(Fig.3);(3)rootsoverexpressinguORF1displayanodule
developmental defect that correlates with reduced
MtHAP2-1 transcript and protein expression (Fig. 4); (4)
transient expression of the uORF1 protein in N. ben-
thamiana leads to a reduction on the level of a GUS
fusion containing the alternatively spliced MtHAP2-1
5(cid:2)LS (Fig. 5); and (5) uORF1p is able to bind both the
alternatively and normally spliced MtHAP2-1 5(cid:2)LS-cor-
respondingmRNAs(Fig.6).
ThisworkdemonstratesthatuORF1pisabletoactin
trans to down-regulate gene expression, although our
data do not allow us to exclude the possibility of addi-
tionalcisregulationmediatedbyMtHAP2-1uORF1.Itis
still unclear how binding of uORF1p to the MtHAP2-1
5(cid:2)LS-corresponding mRNA leads to reduced transcript
accumulation. We can exclude this to be due to transi-
tivesilencingmediatedbythe5(cid:2)LSinN.benthamiana,
becauseexpressionofuORF2,uORF3,oramutatedver-
sion of uORF1 in which the ATG start codon had been
mutated to AGG, does not result in transcript down-
regulation.SinceuORF1pactsintrans,wedonotexpect
down-regulation of MtHAP2-1 expression to be due
to ribosome stalling mediated by the uORF nascent
peptide, in contrast to previously described cis-acting
Figure7. ModelforregulationofMtHAP2-1expressiontoor- uORFs. It is possible that overexpression of uORF1p
chestratenoduledevelopment.(A)Fourdaysafterinoculation, somehow results in blockage of either (1) transcription,
the nodule structure is not well differentiated. miR169 and resultinginreducedaccumulationofMtHAP2-1mRNA,
uORF1pnotbeingexpressed,MtHAP2-1presentsahigherex-
or (2) translation, leading to degradation of a nontrans-
pression level, evenly distributed throughout the nodule (data
lated transcript by NMD or other mechanisms. How-
notshown).(B)Tendaysafterinoculation,miR169isexpressed
ever,thefactthatamutantversionofuORF1palsobinds
inthenoduleinfectionzone(II),leadingtoMtHAP2-1transcript
down-regulation and restricting MtHAP2-1 expression to the the MtHAP2-1 5(cid:2)LS-corresponding mRNA but does not
meristematiczone(I)andnormalnodulegrowth(Combieretal. result in transcript degradation (J.P. Combier and S.
2006).(C)Twenty-onedaysafterinoculation,ASofMtHAP2-1 Rivas,unpubl.)makesthesetwopossibilitieslesslikely.
occurs,resultinginexpressionofuORF1pintheinfectionzone. Alternatively, it is tempting to speculate that uORF1p
uORF1p binding to the MtHAP2-1 transcript induces mayrecruitadditionalproteinsinvolvedinnucleicacid
MtHAP2-1transcriptdegradation,therebyrestrictingMtHAP2-1 degradation. Since uORF1p-dependent mRNA down-
expressiontothemeristematiczoneofthenodule. regulationisactiveinN.benthamiana,itislikelythat,
although BLAST analysis did not identify any uORF1-
duce a cis-acting peptide that causes effector molecule- like gene in other organisms, the protein(s) required for
dependent stalling of the ribosomes at the end of the uORF1p-dependent mRNA down-regulation are con-
uORF.Inothercasesitisthelengthorposition,orother servedamongdifferentplantspecies.
features of the uORF, rather than the encoded peptide, To our knowledge, this is the first report of a uORFp
that determine the efficiency with which ribosomes actingintranstodown-regulategeneexpressionfollow-
reinitiate downstream translation. Translation of the ing AS, and therefore it contributes to a broader under-
uORF can also control gene expression by affecting the standingoftheroleplayedbyuORFsintheregulationof
stabilityofthemRNA(Gabaetal.2005).Finally,trans- geneexpression.Furtherstudiesarenowrequiredtode-
actingfactorsmayparticipateinregulatorymechanisms terminetowhatextentthismechanismcanbegeneral-
(Vilela and Mc Carthy 2003). uORF-mediated transla- izedbeyondthecontrolofMtHAP2-1expressionduring
tional regulation in cis has been reported for several noduledevelopment.
plant genes including Opaque 2 and Lc in maize, and
SAMDC, ATB2, and SAC51 in Arabidopsis (Imai et al.
2006,andreferencestherein). Materialsandmethods
Here we describe a novel mode of action for a uORF.
Themajorfindingstoemergefromourexperimentsare Biologicalmaterial
the following: (1) The regulatory effect of uORF1p on
M. truncatula Gaertn cv. Jemalong genotype A17 was grown
MtHAP2-1 expression is dependent on AS of the first
aeroponically as described (Journet et al. 2001) and inoculated
introninthe5(cid:2)LSofMtHAP2-1;(2)despitethefactthat
with the S. meliloti strain RCR2001 pXLGD4 (GMI6526)
the context of uORF1 start codon (CCTTTTATGA) is (Ardoureletal.1994)asdescribedpreviously(ElYahyaouietal.
notveryfavorable(Kozak2002),initiationoftranslation 2004).Agrobacterium-mediatedtransientexpressioninN.ben-
atuAUG1canbedetectedinthenoduleinfectionzone thamianaleaveswasperformedasdescribed(Rivasetal.2004).
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MtHAP2-1intrans-regulationbyauORF
Root transformations were performed as reported previously aligning at intron 4 of MtHAP2-1, were used to confirm the
(Combieretal.2006). absenceofcontaminatinggenomicDNA.
Anti-MtHAP2-1antibodyproduction
Vectorsandconstructs
Anti-MtHAP2-1polyclonalantibodieswereraisedinrabbitby
PCRamplificationwasperformedusingPfxpolymerase(Invit-
Eurogentec(http://www.eurogentec.be)againsttwopeptidesin
rogen)andusedprimersareshowninSupplementalTableS1.
theNterminusofMtHAP2-1;namely,1MAMQPVYLKEHEGNV15
ForM.truncatularoottransformation,pPEX-derivedvectors
were used (Combier et al. 2006). To generate the RNAiintron1 and64APSKNLVRGVEQLFD78.
construct, a 249-bp sequence from the intron 1 was amplified
using intron1RNAi5(cid:2) and intron1RNAi3(cid:2) primers, and cloned Expressionanalysis
into pPEX RNAi vector. For the uORFs-GUS fusions, 2300-,
Histochemical GUS staining were performed and analyzed as
2373-,and2616-bpsequencesupstreamofuATG1,uATG2,and
described(Combieretal.2006).
uATG3, respectively, were amplified with primers MtHAP2-
1Pro5(cid:2) and uORF1GUS3(cid:2), uORF2GUS3(cid:2), and uORF3GUS3(cid:2), Toanalyzeproteinexpression,rootmaterialorN.bentami-
analeafdiscsweregroundinliquidnitrogenand50–100mgof
respectively, and cloned into a pPEX-GUS vector. The
powderwereresuspendedin100µLofextractionbuffer(50mM
35S:uORF1,35S:uORF2,and35S:uORF3constructsweremade
Tris-HCl at pH 7.4, 150 mM NaCl, 10% glycerol [v/v], 1 mM
after uORF1, uORF2, and uORF3 PCR amplification, with
uORF15(cid:2)anduORF13(cid:2),uORF25(cid:2)anduORF23(cid:2),anduORF35(cid:2) DTT, 1 mM PMSF, 1% plant protease inhibitor cocktail
anduORF33(cid:2)primers,respectively,andcloningintopPEX.For [Sigma])andcentrifugedat10,000gfor10minat4°C.Protein
concentrationinthesupernatantwasdeterminedwiththeBrad-
the MtHAP2-1 overexpression construct, the MtHAP2-1 gene
was amplified with MtHAP2-1 5(cid:2) and MtHAP2-1 3(cid:2)2 primers fordproteinassaykit(Bio-Rad),usingBSAasastandard.Fifty
micrograms of total protein were separated on a NuPage 4%–
andclonedintothepPEXvector.
12%Bis-Trisgels(Invitrogen)accordingtothemanufacturer’s
For the N. benthamiana transient assays, a 35S:ORF1-HA
instructions and transferred onto Protran BA85 nitrocellulose
construct was generated after amplification of uORF1 using
uORF15(cid:2)anduORF13(cid:2)primersandcloningintothepBIN19g membranes(Schleicher&Schuell)bywetelectroblotting(Mini-
Protean II system; Bio-Rad). For detection of MtHAP2-1 and
vector (Rivas et al. 2004). For overexpression of HA-tagged
GUS,theblotswereincubatedwithanti-MtHAP2-1polyclonal
uORF1 mutated in the start codon ATG, we used the uORF1
AGG5(cid:2)primer.TogeneratetheGUSfusiontothealternatively antibodies (1:10,000 dilution) and rabbit anti-(cid:2) glucuronidase
spliced MtHAP2-1 5(cid:2)LS, the 5(cid:2)LS was amplified using 5(cid:2)LS5(cid:2) polyclonalantibodies(1:1,000dilution;FitzgeraldIndustriesIn-
and5(cid:2)LS3(cid:2)andclonedinposition5(cid:2)oftheGUScassetteinthe ternational),respectively.Afterincubationwiththesecondary
antibody (anti-rabbit IgG-peroxidase, 1:15,000; Sigma), bands
pPEXGUSvector.
werevisualizedusingtheECLPluskit(AmershamPharmacia
Biotech)understandardconditions.FordetectionofHA-tagged
Histochemicalstainingandmicroscopystudies uORF1p, blots were incubated with anti-HA rat monoclonal
(cloneEF10)antibodies(Roche),linkedtohorseradish,atafinal
Nodule sections (50 µm thick) were prepared and observed as
dilutionof1:1,000.
described (Combier et al. 2007). Histology of the nodule was
performedonfixedmaterialembeddedinTechnovit7100resin
(HereausKulzer).Four-micronthicksectionswereobservedin RNApull-downassays
darkfieldorepifluorescencemicroscopyafternuclearstaining MtHAP2-1 with no 5(cid:2)LS was amplified by PCR from cDNA
withDAPI(diaminophenylindol).Inallcases,shownfiguresare
fromM.truncatulanodules,4dafterinoculationwithS.me-
representative of three to five independent transformation ex- liloti,usingprimersMtHAP2-15(cid:2)and3(cid:2)LS3(cid:2)andclonedintoa
periments,inwhichabout30transgenicrootswerechosenand
pGEM-Tvector(Promega).ThisconstructwasnamedMtHAP2-
∼10–20individualnoduleswereobserved. 1(cid:3)5(cid:2)LS.
Alternatively spliced MtHAP2-1 5(cid:2)LS was amplified using
primers 5(cid:2)LS5(cid:2) and 5(cid:2)LS3(cid:2) from M. truncatula genomic DNA,
RNAextractionandqRT–PCRanalysis
clonedintopGEM-TandnamedMtHAP2-15(cid:2)LS-AS.
Material for RNA analysis was ground in liquid nitrogen, and Finally, normally spliced MtHAP2-1 was amplified from M.
totalRNAwasisolatedusingtheSVtotalRNAextractionkit truncatulanodules,4dafterinoculationwithS.meliloti,using
(Promega) according to the manufacturer’s recommendations. primers5(cid:2)LS5(cid:2)and3(cid:2)LS3(cid:2)andclonedintopGEM-T.Thiscon-
Reverse transcription was performed using 3 µg of total RNA structwasnamedMtHAP2-1-NS.
andSuperScriptreversetranscriptaseII(Invitrogen).qPCRwas As a control, the coding sequence of the Arabidopsis tran-
runonaLightCyclersystem(RocheDiagnostics)accordingto scription factor AtMYB30 (At3g28910) was also amplified and
themanufacturer’srecommendations.MtEF1(cid:1)orNbEF1(cid:1)were clonedintopGEM-T.
usedasaninternalstandard(ElYahyaouietal.2004).Primers PCR fragments containing either MtHAP2-1(cid:3)5(cid:2)LS,
used in the different experiments are listed in Supplemental MtHAP2-15(cid:2)LS-AS,orMtHAP2-1-NS wereamplifiedusingthe
TableS1.Thespecificityoftheamplificationswasverifiedby M13FwdandM13Revprimers,andusedforinvitrotranscrip-
sequencingofthePCRproductsandbymeltingcurveanalysis tionusingaRiboprobeCombinationSystemSP6/T7(Promega),
at the end of each experiment. Efficiency of the amplification inthepresenceofbiotinylatedUTP.ThebiotinylatedRNAs(or
wasverifiedbytheanalysisofstandardcurves. differentratiosofnonbiotinylatedtobiotinylatedRNAs)were
PrimersuORF1q5(cid:2)anduORF1q3(cid:2)wereusedtoquantifythe boundtostreptavidinmagneticbeads(µMACSStreptavidinKit;
MtHAP2-1 unspliced form, and exon1–2 and MtHAP2-1TP4 Milteny Biotec) for 1 min at room temperature and incubated
primerswereusedforquantificationofthesplicedform.Am- withvariousamountsofatotalproteinextractcontainingHA-
plificationofeitherformwasconfirmedbysequenceanalysisof taggeduORF1pfromN.benthamianaagroinfiltratedleaves,for
theobtainedPCRfragments.Intron45(cid:2)andintron43(cid:2)primers, 1hat4°C,asdescribed(Campalansetal.2004).Boundproteins
GENES&DEVELOPMENT 1557
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Combieretal.
wereelutedinproteinloadingbufferandanalyzedbyWestern Cuperlovic-Culf,M.,Belacel,N.,Culf,A.S.,andOuellette,R.J.
Blot using anti-HA rat monoclonal (clone EF10) anti- 2006.Microarrayanalysisofalternativesplicing.OMICS10:
bodies(Roche)asdescribedabove.NonbiotinylatedMtHAP2-1 344–357.
5(cid:2)LS-AS-correspondingRNAwasalsousedincompetitionex- Di Agostino, S., Strano, S., Emiliozzi, V., Blandino, G., and
perimentstoelutethebounduORF1p. Piaggio,G.2006.Gainoffunctionofmutantp53:Themu-
tant p53/NF-Y protein complex reveals an aberrant tran-
scriptionalmechanismofcellcycleregulation.CancerCell
Accessionnumbers
10:191–202.
TheGenBankaccessionnumberofMtHAP2-1anduORF1pde- ElYahyaoui,F.,Kuster,H.,BenAmor,B.,Hohnjec,N.,Puhler,
scribedinthisstudyisEF488826. A.,Becker,A.,Gouzy,J.,Vernie,T.,Gough,C.,Niebel,A.,et
al.2004.ExpressionprofilinginMedicagotruncatulaiden-
tifies more than 750 genes differentially expressed during
Acknowledgments nodulation,includingmanypotentialregulatorsofthesym-
bioticprogram.PlantPhysiol.136:3159–3176.
WethankMartinCrespiandDavidBarkerforcriticalreadingof
Foucher,F.andKondorosi,E.2000.Cellcycleregulationinthe
themanuscript.ThisworkwassupportedbyFP6GrainLegume
courseofnoduleorganogenesisinMedicago.PlantMol.Biol.
IntegratedProject(FP6-2002-FOOD-1-506223).
43:773–786.
Gaba, A., Jacobson, A., and Sachs, M. 2005. Ribosome occu-
pancy of the yeast CPA1 uORF termination codon modu-
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