Processing of preribosomal RNA in Saccharomyces cerevisiae

19 Pages·2015·0.54 MB·English

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Processing of preribosomal RNA
Saccharomyces cerevisiae
in
Antonio Fernández-Pevida,1,2 Dieter Kressler3 and Jesús de la Cruz1,2∗
Most, if not all RNAs, are transcribed as precursors that require processing to
gainfunctionality.RibosomalRNAs(rRNA)fromallorganismsundergobothexo-
and endonucleolytic processing. Also, in all organisms, rRNA processing occurs
inside large preribosomal particles and is coupled to nucleotide modification,
foldingoftheprecursorrRNA(pre-rRNA),andassemblyoftheribosomalproteins
(r-proteins). In this review, we focus on the processing pathway of pre-rRNAs of
cytoplasmic ribosomes in the yeast Saccharomyces cerevisiae, without doubt, the
organism where this pathway is best characterized. We summarize the current
understanding of the rRNA maturation process, particularly focusing on the
pre-rRNAprocessingsites,theenzymesresponsibleforthecleavageortrimming
reactions and the different mechanisms that monitor and regulate the pathway.
Strikingly, the overall order of the various processing steps is reasonably well
h conserved in eukaryotes, perhaps reflecting common principles for orchestrating
c theconcomitanteventsofpre-rRNAprocessingandribosomeassembly. ©2014John
. Wiley&Sons,Ltd.
o
r
e
r
.
c
o INTRODUCTION at high efficiency and it has been calculated that the
d pre-rRNA transcripts make up more than 60% of all
In practically all eukaryotes, ribosomal DNA
/ nucleartranscripts.1
/ (rDNA) is arranged in two transcriptional units:
: Figure 1(b) outlines the current knowledge on
p the 5S rRNA gene on one hand and the unit con-
thediversestepsofthepre-rRNAprocessingpathways
t taining the 18S, 5.8S, and 25S rRNA genes on the
t other hand. To cope with the high demand for cyto- in yeast. In this part, we provide an overall descrip-
h
tion of these pathways, while detailed discussions of
plasmic ribosomes, the amplification of the copy
the specific steps will be presented in the following
number of rDNA genes has been evolutionarily
selected. Saccharomyces cerevisiae has about 150 sections. The 5S rRNA is transcribed by RNA poly-
rDNA copies per haploid genome on chromosome merase III (RNAPIII) as a precursor whose 5′ end
XII, organized as a tandem array, where, in contrast corresponds to the mature 5′ end, while its 3′ end
to higher eukaryotes, the 5S rDNA gene is a part of is extended by about 12 nucleotides (nt) (Figure 2).
the rDNA repeat unit (Figure 1(a)). Despite the high The 3′ end of the pre-5S rRNA is a U-rich sequence
copynumber,onlyabouthalfoftherDNAcopiesare as RNAPIII generally uses T-rich sequences contigu-
transcribed under exponential growth conditions in ous to the mature 3′ ends of RNAs as a termination
yeast.Notably,theactiverDNAunitsaretranscribed signal.2 As discussed later, the 3′ end of mature 5S
rRNA is generated exonucleolytically (Figure 2). The
∗Correspondenceto:[email protected] 18S,5.8S,and25SrRNAsaretranscribedbyRNAPI
1Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario asasinglepolycistronictranscriptflankedbyexternal
VirgendelRocío/CSIC/UniversidaddeSevilla,Sevilla,Spain transcribed spacers at both ends (5′ and 3′ external
2DepartamentodeGenética,UniversidaddeSevilla,Sevilla,Spain
transcribed spacers (ETS)) and containing two inter-
3Unit of Biochemistry, Department of Biology, University of
nal transcribed spacers (ITS1 and ITS2) that separate
Fribourg,Fribourg,Switzerland
thematurerRNAsequences(Figure1(a)).Thelongest
Conflict of interest: The authors have declared no conflicts of detectable RNAPI precursor, named 35S pre-rRNA,
interestforthisarticle.
begins at a transcription start site, which is placed
1
(a)
(b)
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FIGURE 1|
Pre-rRNAprocessinginS.cerevisiae.(a)StructureofanrDNArepeatunit.Eachunitcontainsalargeelementencoding18S,5.8S,and
25SrRNAs,whichistranscribedbyRNAPI,andashortelementencoding5SrRNA,whichistranscribedbyRNAPIII.Nontranscribed,externaland
internalspacers(NTS,ETS,andITS,respectively)areindicated.ThesequencesforthematurerRNAspeciesareshownasbarsandthespacersaslines
(NTSsareshownthinnerthanETSsorITSs).Transcriptionstartsitesareshownasredarrowsandtranscriptionstopsites(T andT )areshownas
1 5S
smallpurpleboxes.Thedistinctprocessingsitesareindicated.(b)Schemeofthepre-rRNAprocessingpathway,whichisfurtherdescribedinthetext.
Thenucleasesresponsibleoftheendonucleolyticcleavages(red)andexonucleolytictrimmings(green)arehighlighted.Notethatsome
endonucleaseshavenotyetbeenidentified.
2
FIGURE 2|
SecondarystructuremodelforS.cerevisiaepre-5S
rRNA.Thesecondarystructureaswellastheresidueandhelix(H)
numberswastakenfromtheComparativeRNAWebSite
(www.rna.ccbb.utexas.edu/).Notethatformationof3′endofmature
5SrRNAreliesonaRex1-dependent3′–5′exonucleolyticstep.Seetext
forfurtherdetails.
about 700nt upstream of the 5′ end of mature 18S FIGURE 3|SecondarystructuremodelfortheS.cerevisiae3′ETS.
h rRNA.3 Atits3′ end,thisprecursorendsabout14nt Rnt1cleavagesitesareindicated.The3′endofmature25SrRNA(blue)
c downstreamofthe3′ endofmature25SrRNA.4 This isgeneratedbyRex1-dependent3′–5′exonucleasetrimmingfromthe
o. 3′ end is generated by co-transcriptional cleavage of B0site.Seetextforfurtherdetails.
r thenascentRNPAItranscript.4Ithasbeenshownthat
e termination of RNAPI transcription mostly occurs pre-rRNAtranscriptioniscompleted.11Morerecently,
r just upstream of the Reb1-binding site at a T-rich co-transcriptionalcleavageofnascentpre-rRNAtran-
.
c sequence,namedT1,whichisplacedabout90ntfrom scripts at site A2 has been supported by carefully
o the 3′ end of mature 25S rRNA.5 A second termi- analysing the kinetics of pre-rRNA maturation and
nation site, named fail-safe or T2, which is located by visualization of nascent pre-rRNA transcripts
d
further downstream at about 250nt from the 3′ end by electron microscopy (EM).1213 It has been esti-
/
/ of mature 25S rRNA, is used when read-throughs mated that about 70% of pre-rRNAs undergo
:
p occur at T1.5 Most nascent RNAPI transcripts are co-transcriptional cleavage in exponentially growing
t co-transcriptionally cleaved by endonuclease Rnt1 at wild-type yeast cells.12 Co-transcriptional cleavage
t two positions (+14/15 and +49/50 relative to the 3′ appears to occur when the RNAPI has traveled into
h
end of mature 25S rRNA) lying at opposite sides of the 25S rDNA around 1.5kb downstream of site
a stem-loop structure within the 3′ ETS (Figure 3).4,6 A and requires Rrp5 and specific 60S r-subunit
2
Co-transcriptionalRnt1-dependentcleavageisimpor- biogenesisfactors.13–15
tantforefficientterminationatT1,becausethiscleav- Cleavage at site A , either co- or
2
age generates the 5′ end site required for recruit- post-transcriptional, generates the 20S pre-rRNA,
ment of the exonuclease Rat1 containing complex, which is then processed to mature 18S rRNA by
which degrades the remaining nascent transcript to endonucleolytic removal of the D-A fragment
2
stimulatetranscriptionterminationwhenreachingthe (Figure1(b)).Interestingly,bothinyeastandinhigher
paused RNAPI, a process referred to as ‘torpedo- eukaryotes,thisreactionoccursinthecytoplasm.16,17
ing’theRNAPI.5,7,8 WhenRnt1orRat1activitiesare Whyevolutionhasselectedthiscompartmentalization
impaired, RNAPI fails to terminate efficiently at T1 of pre-rRNA processing remains unclear. Cleavage
andmainlystopsatT2.5,7 at site A also generates the 27SA pre-rRNA, which
2 2
Removalofthepre-rRNAspacersisamulti-step is then processed by two alternative pathways that
process; the 35S pre-rRNA is initially cleaved both lead to the formation of mature 5.8S and 25S
at sites A and A in 5′ ETS and at site A in rRNAs (Figure 1(b)). In the major pathway, about
0 1 2
ITS1 (Figure 1(a)), generating the 20S and 27SA 85%of27SA pre-rRNAisfirstcleavedatsiteA by
2 2 3
pre-rRNAs and thus separating the pre-40S and endonucleaseRNaseMRP,whichisaribonucleopro-
pre-60S ribosomal subunits (r-subunits).9,10 In tein(RNP)particlewhoselargeRNAmoietyharbors
the late 80s, experimental evidence indicated that thecatalyticactivity.18Theresulting27SA pre-rRNA
3
removal of the 5′ ETS could take place before 35S is 5′–3′ exonucleolytically digested up to site B to
1S
3
yield the 27SB precursor, which contains the mature directly participate in the nucleolytic events. For
S
5′ endofthe5.8S rRNA.19,20 Intheminorpathway, other aspects of pre-rRNA processing (e.g., role of
S
about 15% of 27SA pre-rRNA is processed directly cis-acting elements within the pre-rRNA sequences),
2
to site B , apparently by an endonucleolytic event. rRNA modifications or ribosome assembly in yeast
1L
This27SB precursorharborsthemature5′endofthe and pre-rRNA processing in higher eukaryotes, we
L
5.8S rRNA,whichislocatedabout6ntupstreamof referreaderstootherreviews.30–39
L
the 5′ end of the 5.8S rRNA.21 It has been reported
S
that almost concurrently with B processing, the
1
3′ end of mature 25S rRNA is generated by 3′–5′ THE INITIAL EVENTS
trimming to site B of the remnant B –B fragment
2 2 0 Co-transcriptional Cleavage at Site B
presentin27SApre-rRNAs.4,22 0
In the post-transcriptional pathway, the earliest
Processing of ITS2 from both 27SB species
pre-rRNA processing event is the endonucleolytic
appearstobeidentical(Figure1(b)).Initially,cleavage
cleavage of the nascent pre-rRNA in the 3′ ETS at
at site C separates 7S and 25.5S pre-rRNAs, which
2 site B , thereby generating the 35S pre-rRNA. This
are the precursors of mature 5.8S and 25S rRNAs, 0
step is performed by the dsRNA endonuclease Rnt1,
respectively.22 Strikingly, the endonuclease that per-
which is homologous to bacterial RNase III.4,6 Rnt1
forms this reaction is still unknown. The around
140nt of ITS2 at the 3′ end of the 7S pre-rRNAs are cleaves both strands of an imperfect stem-loop struc-
ture (Figure 3) capped by an (A/U)GNN tetraloop at
rtoem5o′veexdonbuycalesatseepsw,2i3s–e2m6 wechhiacnhislemadinsvtoolvthinegfsoervmeraatilo3n′ positions+14and+49fromthe3′ endofmature25S
h rRNA4;notably,thisreactioncouldbereproducedin
.c odfiaatetsl,ea5s.t8Stw+o3d0isacnredte6Sanpdres-traRblNeApsr.e-RrRecNenAtliyn,teitrmhaes- vsuitbrostrwatieth.6,r4e0coCmonbsinisatnenttRlyn,t1losasn-odf-afusnycnttihoenticof3′REnTt1S
o been demonstrated that maturation of 6S pre-rRNAs
leads to reduced synthesis of mature 25S rRNAs and
r into mature 5.8S rRNAs occurs in the cytoplasm.27 accumulationof3′ ETScontainingpre-rRNAs.4
e Two mature 5.8S rRNA species (5.8S and 5.8S
L S In the co-transcriptional pathway, cleavage at
r rRNAs) are produced in a ratio of about 1:5. It is
c. unclear if the two forms of 5.8S rRNAs have dis- spirtee-rBR0NaApsp,eaarssstuoggreesleteadsebnyastcheentob2s7eSrAve2docro2u7pSliAng3
o tinctfunctions.However,the25.5Spre-rRNAis5′–3′ betweenB cleavageandITS1processing.41
digested to mature 25S rRNA.20,28 This processing 0
d
reactionmayoccurintwodiscretesteps,assuggested
/
/ by the detection of a stable 25S′ pre-rRNA interme- Cleavages at Sites A , A and A
p: diate, which is extended by a few nt at the 5′ end of Removal of 5′ ETS is a0n ear1ly event2in both the co-
t mature25SrRNA,inwild-typecells.27,29 and the post-transcriptional pathways of pre-rRNA
ht It is important to stress that pre-rRNA pro- processing.ThisinvolvesthecleavagesatsitesA0 and
cessing occurs inside large RNP complexes called A , which have been demonstrated to be endonu-
1
preribosomal particles.30 These particles contain, cleolytic processes as the excised 5′–A and A –A
0 0 1
in addition to the pre-rRNAs and different sets spacer fragments accumulate in strains defective in
of assembled ribosomal proteins (r-proteins), a components of the nuclear exosome complex and
multitude of nonribosomal factors including small its associated RNA helicase Mtr4/Dob1 and in the
nucleolar RNAs (snoRNAs) and trans-acting pro- 5′–3′ exonucleases Rat1 and Xrn1, respectively.42–44
teins. Loss-of-function of most of these factors leads Cleavage at site A , which is dispensable,31 was
0
topre-rRNAprocessingdefects;thus,withtheexcep- initially identified as a primer stop that specifically
tionofnucleases,itisnotobvioustorecognizewhich decreased on depletion of the U3 snoRNA.9 Site A
1
specific factors are directly involved in the pre-rRNA is the mature 5′ end of 18S rRNA. It is assumed
processing reactions. Less than a dozen nucleases or that, although occurring practically simultaneously,
putativenucleaseshavebeendescribedtobeinvolved cleavage at site A precedes that at site A . However,
0 1
in pre-rRNA processing, and for only few of these, phenotypic analyses of cleavage-sequence and many
an enzymatic activity has been demonstrated. More- trans-acting factor mutants clearly indicate that pro-
over, bona fide co-factors for nucleases have only cessingatthesesitesisnotcoupled.Thus,cleavageat
scarcelybeenreported.Finally,onlyfewstepsofyeast siteA isnotaprerequisiteforcleavageatsiteA and
0 1
pre-rRNA processing could so far be reproduced in vice versa. In contrast, cleavage at site A is coupled
1
vitro. This review summarizes our current under- to that at site A , and although cleavage at site A
2 1
standing of yeast pre-rRNA processing, particularly couldoccurwithoutsimultaneouscleavageatsiteA ,
2
focusing on the role of the trans-acting factors that it has so far not been reported that cleavage at site
4
A couldtakeplacewithoutpriorcleavageatsiteA . mutations in RCL1 that impair in vivo cleavage at
2 1
CleavageatsiteA isalsoanendonucleolyticeventas site A also abolish pre-rRNA cleavage in vitro.49
2 2
theexcisedD–A andA –A fragmentsaredetectedin From these studies, it has also been deduced that
2 2 3
wild-typecellsandalsoaccumulateinstrainsdefective Rcl1 recognizes a single-stranded substrate, which is
in the 5′–3′ exonucleases Rat1 and Xrn1.43 Among consistent with the proposed location of site A in
2
thepre-rRNAprocessingreactions,cleavageatsiteA the ITS1 secondary structure38 (Figure 5). Interest-
2
isspecialsinceitdefinesthesiteforco-transcriptional ingly, recent data indicate that the trans-acting factor
cleavage and notably separates the intermediates of Rrp5 binds to an evolutionarily conserved stem-loop
thelargeandsmallr-subunitsynthesispathways. located3′ adjacenttositeA inITS1,suggestingthat
2
Processing at sites A , A and A occurs within Rrp5 could define the exact position where cleavage
0 1 2
a large preribosome intermediate known as SSU occurs.15 The SSU processome subunit Utp24, which
processome or 90S preribosomal particle.31 The SSU is predicted to be a nuclease, has been suggested to
processome is a multi-subunit complex composed be the enzyme responsible for pre-rRNA cleavages at
of several snoRNPs (U3, U14, snR30 and snR10) sitesA andA 51 but,invitroexperimentalevidence
1 2,
and multiple trans-acting factors that associate in a is still missing. However, it is worth mentioning that
hierarchical and stepwise manner onto the nascent mutation of residues that are theoretically critical
pre-rRNA.45 Mutation or depletion of any of these for the putative enzymatic activity of Utp24 impairs
subunits usually delays or inhibits cleavage at the cleavage at sites A1 and A2 but not at site A0.51 No
above sites and results in the accumulation of aber- candidate endonuclease for cleavage at site A0 has so
rant 23S, 22S, or 21S pre-rRNAs that extend from farbeenidentifiedanditisevenpossiblethatdistinct
h
+1,A ,orA tositeA ,respectively.31Althoughsome snoRNAs,suchasU3,couldassistinanautocatalytic
c produ0ctive p1rocessing3 from all these intermediates cleavagereactionatthissite.
.
o may occur in wild-type cells, this does not appear to
be the case in processome subunit mutants where a
r FORMATION OF 18S rRNAs
e net deficit in 18S rRNA levels is observed.31 It has
r been demonstrated that the U3 snoRNP, via specific Cleavage at Site D
.
c base-pair interactions between the U3 snoRNA and Pre-rRNA processing at sites A –A releases the
o regions of the 5′ ETS and the 5′ end of mature 18S 20S pre-rRNA, which is rapidly e0xpo2rted from the
d rRNAwithinpre-rRNAs(Figure4),playsanessential nucle(ol)us to the cytoplasm and subsequently pro-
/ role in the assembly of the SSU processome and the cessedatsiteD,the3′endofthemature18SrRNA.16
/ compaction and folding of the nascent pre-rRNA SiteDislikelyasingle-strandedregion52,53(Figure5).
:
p (i.e.,formationofthecentral18SrRNApseudoknot), Processing at site D is endonucleolytic as the D-A
2
t which is a prerequisite for the cleavage reactions to fragment strongly accumulates in yeast mutants
ht occur.47 Interestingly, the U3 snoRNA is unusual in lacking the 5′–3′ exonuclease Xrn1.54 Experimental
that its loss-of-function leads to a strong inhibition evidence indicates that the evolutionarily conserved
ofprocessingatsiteA0,whiledepletionofotherpro- Nob1 protein is the D-site endonuclease. First, Nob1
cessome subunits only results in a modest to severe is a component of cytoplasmic pre-40S particles
kineticdelayofthisprocessingreaction.31 whoseloss-of-functionleadstoastrongaccumulation
ThelinkofcleavagesatthesitesA ,A ,andA of 20S pre-rRNA and a concomitant depletion of
0 1 2,
especiallythetightcouplingbetweencleavagesatsites mature18SrRNA.55 Moreover,purifiedrecombinant
A andA ,suggeststheappealinghypothesisthatthey Nob1 binds in vitro transcribed pre-rRNA fragments
1 2
might be performed by the same endonuclease. Only containing site D, possibly as a tetramer, and in vivo
few nucleases have been proposed to carry out the Nob1protectsanumberofpre-rRNAsites,including
cleavages at these sites. For example, the conserved, site D and its adjacent nts.52,56 Most importantly,
essential protein Rcl1 is required for in vivo 18S Nob1, purified from yeast, cleaves in vitro an RNA
rRNA synthesis.48 As occurs for many components oligonucleotide containing the RNA sequence sur-
of the SSU processome, inactivation of Rcl1 leads to rounding site D precisely at this position.57 This in
inhibition of processing at sites A , A , and A , with vitro assay is dependent on the presence of Mn2+, an
0 1 2
processing at sites A and A being less affected than ioncofactorofPIN-domainnucleases.Theenzymatic
0 1
that at site A 48,49 More recently, it has been shown activity is however very poor and requires elevated
2.
that purified recombinant Rcl1 cleaves in vitro tran- concentrations of protein, Mn2+ and RNA substrate
scribed pre-rRNA fragments that include the 3′ end and long incubation times57; this suggests that some
ofmature18SrRNAandITS1,atasitethatcoincides important cis-elements for cleavage are not present
withtheinvivomappedsiteA 49 Interestingly,point on the minimal RNA substrate and/or that protein
2.
5
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c FIGURE 4|SecondarystructuremodelfortheS.cerevisiae5′ETS.TherepresentationhasbeenadaptedfromthatproposedbyYehandLee.46
o
ThepositionsofthesitesA andA ,whicharecleavedendonucleolyticallyareindicated.Notethattheendonuclease(s)responsibleofthese
0 1
d cleavagesarecurrentlyunknown.SitesbindingtotheU3snoRNAandsuspectedtobindtosnR10arealsohighlighted(shadedencirclednucleotides
/ ingreenorred,respectively).
/
:
p
co-factors,whichmayassistNob1activityinvivo,are the cytoplasm.60 It has therefore been proposed that
t
t lacking in the assay. In line with the latter possibility, yet another kind of structural rearrangement might
h
it has been shown that Pno1/Dim2 binds directly to occur within cytoplasmic pre-40S particles in order
Nob1,therebyincreasingtheRNAbindingaffinityof to move Nob1 from its primary binding site, appar-
Nob1 in vitro.58 In addition, Ltv1, Prp43, and Pfa1 ently located at the base of helix H40 in 18S rRNA,
interact genetically and functionally with Nob1,57 towardsiteDandunleashitscatalyticactivity.60Other
andmutationofseveralotherlatetrans-actingfactors morespeculativemodelsforNob1activationpropose
and 40S r-subunit proteins impairs cleavage of 20S that D-site cleavage is only enabled on restructuring
pre-rRNA.35 of the long helix H44, which is located close to 3′
Nob1isalreadypresentonnucleolarpreriboso- endof18SrRNA,bytheRNAhelicasePrp43andits
mal particles55; therefore, its cleavage activity must co-factorPfa157 orrequiresassemblyfactorstobring
be prevented until it reaches the cytoplasm. Several Nob1 in proximity of its substrate.58 Recently, it has
mechanisms have been proposed to explain this reg- been shown that 20S pre-rRNA processing requires
ulation of Nob1 activity. First, it has been suggested cytoplasmicinteractionof60Sr-subunitswithpre-40S
that D-site cleavage is promoted by a structural rear- particlesand,moreover,dependsontheGTPaseactiv-
rangementonpreribosomalparticlesinvolvingaRNA ityofthetranslationinitiationfactoreIF5Bandayet
conformationalswitchthatonlyoccursaftercleavage to be identified ATP-binding protein, which is likely
atsiteA inthenucleolus.59 Thisswitchwouldallow the ATPase Rio1.60–62 This scenario has been further
2
therepositioningofNob1tothesiteDcontext,lead- corroborated by the finding that a specific mutation
ing to its catalytic activation. Although not refuted, in the 60S r-subunit protein L3 partially impairs 20S
thismodelhasrecentlybeenquestionedasitdoesnot pre-rRNA processing by impeding Nob1 stimulation
sufficientlyexplaintheexclusiveactivationofNob1in bytheGTPaseactivityofeIF5B.63
6
h
c
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o
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FIGURE 5|SecondarystructuremodelfortheS.cerevisiaeITS1.TherepresentationhasbeenadaptedfromthatproposedbyYehetal.50The
o
positionsofthesitesA ,A ,andDareshown,aswellastheendonucleasesinvolvedintherespectivecleavages.ThepositionsofthesitesB and
d 2 3 1L
B arealsoshown.NotethatwhileprocessingfromsiteA toB occursexonucleolyticallybyareactionperformedbytheredundantexonucleases
1S 3 1S
/ Rat1,Rrp17,orXrn1,cleavageatsiteB occursendonucleolyticallybyaso-farunknownendonuclease.Seetextforfurtherdetails.
/ 1L
:
p
t FORMATION OF 5.8S AND 25S rRNAs such a long-range coupling of processing sites, which
t
are separated by about 4000nt, is mechanistically
h ′
5 End Maturation of 5.8S rRNA:
achieved is still unknown. It can, however, be spec-
Processing at Sites A and B Versus ulatedthatthiscouplingcouldbemediatedbybridg-
3 1S
Processing at Site B ingtrans-actingfactorssuchasRrp5,whichisableto
1L
The specific processing pathway for synthesis of bindtopre-rRNAregionsinITS1andthe3′ terminal
mature 25S and 5.8S rRNA commences 3′ from site partof25SrRNA,15 andr-proteinssuchasL3,which
A attwoalternativesites,A orB (Figure1(b))and associatesearlywithpre-60Sr-particlesbutpredomi-
fin2allyyieldstwodifferentm3ature15L.8SrRNAspecies, nantlybindsto the3′ terminalpartof25SrRNA. As
the 5.8S and the 5.8S rRNAs, which are produced previouslysuggestedbyothers,31,41 couplingbetween
L S
in wild-type cells at a ratio of about 1:5.31 The exis- processing at sites A and B could serve as a qual-
3 0
tenceofheterogeneous5.8SrRNAsisaconstantinthe ity control mechanism that prevents futile processing
eukaryotic kingdom19; however, its biological signifi- fromaberranttruncatedpre-rRNAs.
canceisstillunclear. CleavageatsiteA isdependentontheendonu-
3
Processing at the site A is an endonucleolytic clease RNase MRP in vivo.31,33,65 RNase MRP
3
event as a discrete excised A –A fragment could consists of one RNA molecule and nine protein com-
2 3
be detected in yeast mutants defective in the 5′–3′ ponents, with eight of these being shared with the
exonuclease Rat1.43,64 Cleavage at A likely occurs related endonuclease RNase P.66 Depletion of the
3
post-transcriptionally, as suggested by the observa- RNA constituent or most of the individual protein
tion that deletions in the 3′-ETS region, compris- components of RNase MRP leads to inhibition of
ing the stem-loop structure where cleavage at site B cleavage at site A . As a consequence, only the 5.8S
0 3 L
occurs, specifically impair cleavage at site A 41 How rRNA,butnotthe5.8S rRNA,issynthesized.18,66,67
3. S
7
In addition, two other trans-acting factors have 5.8S rRNA species is further increased in xrn1Δ rat1
been described as specifically participating in cleav- double mutants.7,19,43 As a consequence of impaired
age at site A , the above-mentioned Rrp5 and the of 5.8S rRNA production, a switch in the ratio of
3 S
RNA helicase Dbp3.15,68,69 Only the N-terminal S1 5.8S :5.8S rRNAs is observed (e.g., see20). Strik-
L S
RNA-bindingdomainsofRrp5areessentialforcleav- ingly,some5.8S rRNAisstillproducedinthexrn1Δ
S
age at site A 70 Interestingly, this N-terminal part rat1 double mutant, which suggested the involve-
3.
binds to the RNA component of RNase MRP and ment of another 5′–3′ exonuclease in 5′ end matu-
to a short U-rich pre-rRNA region located few nt 3′ ration of 5.8S rRNA and resulted in the identifica-
S
to cleavage site A 15 strongly suggesting that Rrp5 tion of Rrp17.20 On simultaneous depletion of Rat1
3,
is directly required for cleavage at site A . Further and Rrp17 in an xrn1Δ background, practically no
3
experiments, however, have shown that this require- 5.8S rRNA is produced.20 Rat1 and Rrp17 are pre-
S
mentdoesnotinvolvetherecruitmentofRNaseMRP dominantly nucleolar proteins while Xrn1 is mainly
to pre-60S r-particles, because RNase MRP is appar- cytoplasmic.20,75 Targeting Xrn1 to the nucleus com-
ently still able to bind to pre-60S r-particles on Rrp5 plementsthethermo-sensitivegrowthdefectofarat1
depletion.15 Deletion of the DPB3 gene only kineti- allele, indicating that Rat1 and Xrn1 are function-
callydelaysprocessingatsiteA anddoesnotalterthe allyinterchangeableproteinsthatactindifferentsub-
3
5.8S :5.8S rRNAsratio.68 Consideringitsenzymatic cellular locations.75 Whether overexpression of Rat1
S L
activity, it has been speculated that Dbp3 might opti- or nuclear-targeted Xrn1 is able to complement the
mize the recruitment or the activity of RNase MRP functionofdefectiverrp17allelesremainstobedeter-
bychaperoningthestructureoftheITS1regioninthe mined.Moreover,Rat1interactsphysicallyandfunc-
h
vicinity of site A 68,71 Unfortunately, this conjecture tionally with its nonessential co-factor Rai1.76 The
c 3.
stillrequirestobeexperimentallyvalidated. absenceofRai1stronglyenhancesthedefectsofRat1
.
o Endonucleolytic cleavage at site A has been depletionin5.8S rRNAproduction7;however,itonly
3 S
reproduced in vitro using purified RNase MRP mildlyincreasesthoseofRrp17depletion,20,suggest-
r
e from yeast and short in vitro transcribed RNA ing that Rat1-Rai1 and Rrp17 work independently
r substrates.72,73 TheminimalRNAsubstrateofRNase of each other.20 At present, it is not clear whether
.
c MRP is single-stranded, and all information required the different exonucleases display distinct sequence
o for substrate recognition and site-specific cleavage and/or structural preferences in the A3–B1S fragment
in vitro is included in a region encompassing few nt orwhethertheyactinanentirelyindependentorcoor-
d
upstream and downstream of site A , with the pres- dinatedmanner.
/ 3
/ ence of a cytosine at position +4 being a prerequisite In addition to these three exonucleases,
:
p forthecleavagereactiontotakeplace.73 pre-rRNA processing from site A to site B
3 1S
t Cleavage at site A3 provides the entry site for requires several other trans-acting factors, gener-
t a 5′–3′ exonuclease activity that trims the 76nt in ally known as ‘A factors’, including at least Nop7,
h 3
between sites A and B to generate the 5′ end of Erb1,Ytm1,Rlp7,Nop15,Nsa3/Cic1,Rrp1,Nop12,
3 1S
the 27SB pre-rRNA and hence that of the mature and Has1.77–80 Interestingly, most A factors are
S 3
5.8S rRNA. Three trans-acting factors have been interdependent for their association with preriboso-
S
shown to be involved in this processing step, the mal particles.77 Loss of any of these factors leads to
homologous Rat1 and Xrn1 as well as the unre- accumulation of the 27SA pre-rRNA and reduced
3
lated Rrp17 protein.19,20 These three proteins exhibit synthesisof27SB pre-rRNAandmature5.8S rRNA,
S S
processive hydrolytic 5′–3′ exonuclease activity in whilelevelsofthe27SB pre-rRNAandmature5.8S
L L
vitro,20,74 which is required for the function of these rRNA are not affected.77,78 Despite their function in
factorsinpre-rRNAprocessing.7,20Instrainscarrying A –B processing in ITS1, most A factors bind to
3 1S 3
loss-of-functionmutationsinanyofthecorresponding sites around the 3′ end of 5.8S rRNA, the 5′ end of
RAT1,XRN1,orRRP17genes,longerformsof5.8S 25S, and the 5′ end of ITS2.78 Moreover, only the
rRNA that extend to site A clearly accumulate.19,20 recruitment of Rrp17 to preribosomal particles but
3
The role of Xrn1 and Rat1 in this step is partially not that of Rat1 or Xrn1 appears to be dependent
redundant,however,thecontributionofRat1appears on the presence of A factors.77 In agreement with
3
to be more relevant than that of Xrn1. While muta- this, the analysis of its binding sites in ITS1 revealed
tion in or depletion of Rat1 leads to a significant that Rat1 is bound to regions immediately 5′ to site
accumulation of 5′ extended 5.8S rRNA, only very A and may therefore already be, prior to cleavage
3
low levels of these aberrant species can be detected at site A , present in early pre-60S r-particles.78 It
3
in the xrn1 null mutant. In agreement with a role for is noteworthy that loss of A factors impairs stable
3
both Rat1 and Xrn1, accumulation of 5′ extended assembly of four selected r-proteins L17, L26, L35,
8
and L37, which are adjacent to each other in mature analysis of the 3′ terminal region of ITS1 show that
60S r-subunits and bind to 5.8S rRNAs.77,79 It has thisendonucleaseisnotsequencespecificanddoesnot
beensuggestedthattheA factorsdrivethepre-rRNA dependonsequenceelementsinthe5′adjacentregion
3
rearrangements required to enable 5′ end processing ofITS1.21,38
of the 5.8S rRNA and assembly of r-proteins L17,
S
L26, L35, and L37, which in turn establishes the
proper 27SB pre-rRNA conformation for further Cleavage at Site C
2
ITS2 processing.77–79,81 Notably, L17 binds to helix Removal of ITS2 starts by cleavage at site C , which
2
H2indomainIof25S/5.8SrRNA,formedbetweennt separatesthespecificprecursorsalongthematuration
406-417ofmature25Sandthe5′ endofmature5.8S pathwayof5.8Sand25SrRNA.Thisstepisendonu-
rRNA. It has been put forward that binding of L17 cleolyticasanA –C fragmentclearlyaccumulateson
to helix H2 may act as a barrier that stops the 5′–3′ inactivationofe2xoso2mecomplexsubunitsoritsRNA
exonucleases precisely at the B site.19,77 Formation helicase co-factor Mtr4/Dob1.82,83 Mutational anal-
1S
ofhelixH2requiresamajorpre-rRNArearrangement ysis has demonstrated that cleavage at site C relies
2
where the 5′ end sequence of 5.8S rRNA switches on some structural cis-elements within ITS2, among
from its base-pairing with the distal part of ITS1 to them, the so-called ITS2-proximal stem, which is a
a new base-pairing with 25S rRNA (Figure 5), which structure formed by the base-pairing between the 3′
might be stabilized by L17 binding.39 On depletion end of 5.8S and the 5′ end of 25S rRNAs (Figure 6),
of L17, there is a specific accumulation of pre-rRNA and the nt downstream of the cleavage site.38 In fact,
species whose 5′ ends are about 10nt downstream twobase-pairingsjustcontiguoustositeC arestrictly
h 2
of the B site, thus, at the end of helix H2.77 This required for cleavage, perhaps because these are part
c 1S
shorter pre-rRNA is likely the result of the inability of the recognition site of the specific C -site endonu-
. 2
o of Rat1 to stop at the B site in the absence of L17. clease,whichstillremainstobeidentified.
1S
r Consistently, these aberrant pre-rRNA species accu- In wild-type cells, processing at the site C2 fol-
e mulate only very mildly on inactivation of Rat1 in a lows removal of ITS1; however, the identification
r L17-depleted strain.77 As proposed by others, such a of an A –C fragment indicates that in certain cir-
. 2 2
c mechanismtoblockaneventofpre-rRNAprocessing cumstances, 27SA2 pre-rRNA could be prematurely
o has the advantage that, in conditions of incorrect cleaved at site C2.82 A model to explain this specific
r-protein assembly, the exonuclease responsible for order of processing, which is based on the confor-
d
the pre-rRNA processing would initiate degradation mational status of ITS286 and the sequential associ-
/
/ ofthese,aberrantlyprocessedpre-rRNAs.19,77 ation of trans-acting factors required for ITS1 and
p: As 27SA pre-rRNA molecules can be directly ITS2processing,80,87 hasbeenproposed.Accordingly,
2
t processed at site B1L, thereby generating the 27SBL two alternative, predicted structures appear to exist
t pre-rRNA,31neitherprocessingatsitesA andB nor for yeast ITS2 (Figure 6(a) and (b)), the so-called
h 3 1S
RNase MRP or Rat1 are essential for ribosome bio- ‘ring’ and ‘hairpin’ structures, which differ primar-
genesis.Indeed,anrDNAconstructcontainingalarge ily in base-pairing of the sequences adjacent to the
distaldeletionofITS1encompassingsiteA stillsup- mature 5.8S and 25S rRNA ends.84–86 The transi-
3
portsgrowth,althoughatareducedrate.19,38 Inthese tion from the ring to the hairpin structure is likely
circumstances, the 27SA molecules are only pro- a prerequisite to induce cleavage at site C 86 Appar-
2 2.
cessed at site B and the 5.8S :5.8S ratio increases ently, the A factors, which bind at or near ITS2 (see
1L L S 3
dramatically.19,67 The mechanism by which process- previous section), have different roles in this struc-
ing at site B takes place has not yet been addressed tural transition. While factors such as Nop15 and
1L
in detail. However, three facts can be highlighted: (1) Nsa3 have been suggested to hold ITS2 in the ring
AsproposedforcleavageatsiteA ,processingatsite conformation,77,78 others, such as Rlp7, Nop12, and
3
B appears to occur post-transcriptionally because Pwp1, appear to be important for the formation of
1L
deletions in the 3′ ETS also affect this processing theITS2-proximalstem.80,88,89 Moreover,Nop12and
reaction.41 (2) Processing at site B is likely endonu- Pwp1haveadditionalrolesinpromotingproperfold-
1L
cleolytic, as deduced by the identification of discrete ing of the 5.8S rRNA.80 As discussed above, the
amountsofanA –B fragmentinstrainsdefectivein concerted action of the A factors promotes, either
2 1L 3
the RNA component of RNase MRP or Rrp5.21 As directly or indirectly, the proper assembly of a set of
thisprocessingactivityinvolvesasingleevent,forma- r-proteins(L17,L26,L35,andL37),whichinturnis
tionof5.8S rRNAiskineticallyfasterthansynthesis aprerequisiteforthecorrectassociationofNsa2and
L
of 5.8S rRNA.19 (3) The endonuclease responsible Nog2,whichbelongtotheso-called‘Bfactors’.81,87,90
S
ofthiscleavagereactionisstillunknown.Mutational The B factors include RNA-binding proteins (Nip7,
9
(a) (b)
h
c
.
o
r
e
r
.
c FIGURE 6|SecondarystructuremodelsfortheS.cerevisiaeITS2.TwoconformationshavebeenproposedforITS2,(a)theringconformation84
o and(b)thehairpinconformation.85ThepositionsofthesitesC ,C ′,C ,Easwellasthe3′endextended5.8S+30,6S(+8)and5.8S+5speciesare
1 1 2
d indicated.NotethatthenucleaseresponsibleforendonucleolyticcleavageatsiteC remainstobeidentified.ProcessingfromsiteC tositeC occurs
2 2 1
/ by5′–3′trimmingbyeitherRat1,Rrp17,orXrn1.ProcessingfromsiteC2tositeEoccursby3′–5′trimmingbytheconcertedactionofdifferent
/ exonucleases.Notethatalltheseprocessingreactionarerepresentedonlyinthehairpinstructure.Seetextforfurtherdetails.
:
p
tt Nsa2, Rlp24, Rpf2, and Tif6), a scaffolding protein multi-step process,91 which is performed by different
h (Mak11), GTPases (Nog1 and Nog2), rRNA methyl- nucleases that act in a concerted manner. The reason
transferases (Nop2 and Spb1), and RNA helicases for this complexity is unclear, especially if one con-
(Drs1andHas1),butnoneofthesecanbesuspectedto siders that aberrant 60S r-subunits containing 3′ end
actastheC2-siteendonuclease.81 Loss-of-functionof extended forms of 5.8S rRNAs are translationally
theseBfactorsleadstoacommonpre-rRNAprocess- competent.23,25
ing phenotype, which consists in the accumulation of Four distinct and successive steps during the 3′
27SBpre-rRNArelativeto27SAand7Spre-rRNAs.81
end formation of the 5.8S rRNA can be discerned.
It has been shown that B factors associate sequen- The initial step consists in the 3′–5′ trimming of the
tiallywithpre-60Sr-particlesthroughtwoconverging 7Spre-rRNAs,whichareextendedattheir3′ endsby
pathwaysthatresultinthefinalrecruitmentofNog2. about 140nt up to site C in ITS2, to the 5.8S+30
Whether the GTPase Nog2 promotes the recruitment 2
pre-rRNAspeciesthatstillretainabout30ntofITS2.
orfacilitatestheenzymaticfunctionofthemysterious In the second step, the 5.8S+30 pre-rRNAs are pro-
C2-siteendonucleaseiscompletelyunknown. cessed to 6S pre-rRNAs, whose 3′ ends still contain
about 8nt of ITS2. Both steps are carried out by the
′
3 End Maturation of 5.8S rRNA nuclear exosome. This is a conserved multi-protein
Cleavage at site C releases the 7S and 25.5S complex composed of a core of nine subunits; six
2
pre-rRNAs, which are the immediate precursors of these (Mtr3, Rrp41/Ski6, Rrp42, Rrp43, Rrp45,
of mature 5.8S and 25S rRNAs, respectively. Forma- and Rrp46) harbor an RNase PH fold and form a
tion of the 3′ end of mature 5.8S rRNAs from the 7S hexameric barrel-like structure that is stabilized by
pre-rRNAsisaverycomplicated3′–5′ exonucleolytic a cap formed by three proteins (Csl4/Ski4, Rrp4,
10