Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and ...

14 Pages·2013·2.98 MB·English

Transcriptome-Wide Mapping of 5-methylcytidine RNA
Modifications in Bacteria, Archaea, and Yeast Reveals
5
m C within Archaeal mRNAs
Sarit Edelheit1., Schraga Schwartz2., Maxwell R. Mumbach2, Omri Wurtzel1¤, Rotem Sorek1*
1DepartmentofMolecularGenetics,WeizmannInstituteofScience,Rehovot,Israel,2BroadInstituteofMITandHarvard,Cambridge,Massachusetts,UnitedStatesof
America
Abstract
Thepresenceof5-methylcytidine(m5C)intRNAandrRNAmoleculesofawidevarietyoforganismswasfirstobservedmore
than40yearsago.However,detectionofthismodificationwaslimitedtospecific,abundant,RNAspecies,duetotheusage
oflow-throughputmethods.Toobtainahighresolution,systematic,andcomprehensivetranscriptome-wide overviewof
m5Cacrossthethreedomainsoflife,weusedbisulfitetreatmentontotalRNAfrombothgrampositive(B.subtilis)andgram
negative (E. coli) bacteria, an archaeon (S. solfataricus) and a eukaryote (S. cerevisiae), followed by massively parallel
sequencing.Wewereabletorecovermostpreviouslydocumentedm5CsitesonrRNAinthefourorganisms,andidentified
severalnovelsitesinyeastandarchaealrRNAs.Ouranalysesalsoallowedquantificationofmethylatedm5Cpositionsin64
tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms.
Molecules of tRNAs in which m5C was absent were also discovered. Intriguingly, we detected m5C sites within archaeal
mRNAs,andidentifiedaconsensusmotifofAUCGANGUthatdirectsmethylationinS.solfataricus.Ourresults,whichwere
validated using m5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea,
suggestingthat thismode ofpost-transcriptional regulation extends beyond the eukaryotic domain.
Citation:EdelheitS,SchwartzS,MumbachMR,WurtzelO,SorekR(2013)Transcriptome-WideMappingof5-methylcytidineRNAModificationsinBacteria,
Archaea,andYeastRevealsm5CwithinArchaealmRNAs.PLoSGenet9(6):e1003602.doi:10.1371/journal.pgen.1003602
Editor:ValeriedeCre´cy-Lagard,UniversityofFlorida,UnitedStatesofAmerica
ReceivedSeptember30,2012;AcceptedMay17,2013;PublishedJune27,2013
Copyright:(cid:2)2013Edelheitetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:RSwassupported,inpart,bytheEuropeanResearchCouncil(StartingGrant260432),theMinervaFoundation,theIsraeliScienceFoundation(grant
ISF-1303/12),theLeonaM.andHarryB.HelmsleyCharitableTrust,andtheDeutscheForschungsgemeinschaft(aDIPgrant).Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
.Theseauthorscontributedequallytothiswork.
¤Currentaddress:WhiteheadInstitute,MIT,Cambridge,Massachusetts,UnitedStatesofAmerica.
Introduction responses to several stress conditions, suggesting that these
modifications may play a role in cellular response to stress,
5-methylcytidine (m5C) is a modification that occurs both on potentially by mediating translation rates [8,9]. In rRNA m5C is
DNA and RNA. In eukaryotes, this DNA modification has been thoughttoplayaroleintranslationalfidelity[10].
extensively studied over the past years, and was found to play a Traditional methods for studying m5C and other RNA
crucial role in genomic imprinting, X-chromosome inactivation, modifications include HPLC and mass spectrometry, requiring
and suppression of repetitive elements [1,2].Less isknown about isolationofthespecificRNAmoleculebeingstudiedtonearpurity
the distribution and role of m5C sites in RNA. In bacteria, m5C [11,12]. As opposed to the highly abundant tRNAs and rRNAs,
positions were described only in rRNA, whereas in archaea and isolation of specific mRNA molecules to purity is experimentally
eukaryotesm5CwasmappedtobothtRNAandrRNA.IntRNA challenging, and hence studies of m5C modifications on mRNA
molecules, m5C sites are typically present at the variable region molecules were limited until recently. Indeed, early studies
andtheanticodonloop,wheretheyhavebeenshowntostabilize documenting the presence of m5C in mRNA have been
the secondary structure of the tRNA and affect codon identifica- controversial;somestudieshavefailedtodetectm5Cineukaryotic
tion and tRNA aminoacylation [3–5]. For example, m5C at mRNA [13–16], whereas others have identified this modification
position 40 of the yeast tRNA-Phe enables conformational [17–19].
transition of the entire anticodon loop, inducing Mg2+ binding at Recently, the presence of m5C in human HeLa mRNA was
adistantsiteandresultinginstructuralstabilization[6].Absenceof studiedinaglobalmanner,identifyingthousandsofpotentialm5C
anm5Cmodificationintheanticodonwobble-base(position34)of siteswithinhumanmRNA[20].ThisstudyutilizedRNAbisulfite
tRNA-Leu in yeast was associated with decreased functionality, treatment,whichselectivelyconvertsCresidues,butnotm5C,into
observedwhennonsensesuppressorfunctionwasestimatedinvivo U residues [21], followed by whole transcriptome sequencing.
[7]. Recently, it was shown that various modifications on tRNA, Such bisulfite treatment is commonly used on DNA sequences
including m5C, are dynamically modulated during cellular when DNA methylation states are studied [22,23]. However, its
PLOSGenetics | www.plosgenetics.org 1 June2013 | Volume 9 | Issue 6 | e1003602
m5CRNAModificationinthe3DomainsofLife
coverage ($5 reads) for all rRNA molecules annotated for the
Author Summary
organisms tested, and for 153 of the 187 (82%) annotated tRNA
Ribonucleic acids are universally used to express genetic molecules(Table 1).
information in the form of gene transcripts. Although we Toidentifyputativemethylatedpositions,wealignedthereads
envision RNA as a mere copy of the DNA four-base code, against genomic DNA that was computationally adjusted so that
modification of specific RNA bases can expand the all C residues were converted to T. Overall, we observed that
information code. Such modifications are abundant in .98% of all sequenced C residues were converted into T
transferRNA(tRNA)andribosomalRNA(rRNA),wherethey following bisulfite conversion and cDNA sequencing, providing
contribute to translation fidelity and ribosome assembly.
statistical power to identify cytosine residues that reproducibly
Recent studies in eukaryotes have shown that mRNA failed to undergo conversion as putative m5C sites (Table 1). For
modifications such as RNA-editing (conversion of an
each genomic cytosine, we calculated the number of reads in
adenosine base to inosine), N6-adenine methylation
which that C did not undergo conversion to T, indicative of
(m6A),and5-methylcytidine(m5C)canchangethecoding
methylation(Figure1).Foreachsuchposition,wedeterminedits
sequence, alter splicing patterns, or change RNA stability.
‘‘methylationlevel’’,correspondingtotheproportionoftranscripts
However, no mRNA modifications in bacteria or archaea
in which that position was methylated, and calculated a p-value
have been documented to date. We have used an
approachthatenablesmappingofthem5Cmodifications based on Fisher’s exact test against the null hypothesis that the
across all expressed genes in a given organism. Applying position was not methylated (Methods). Based on previous
this approach on model bacterial, archaeal, and fungal experiments using bisulfite sequencing of DNA [24] and RNA
microorganisms enabled us to reveal the modified RNA [20], where stretches of non-converted Cs were often found in
basesintheseorganisms,andtoprovideanaccurateand specific reads (presumably reflecting insufficient exposure of a
sensitive map of these modifications. In archaea, we transcripttobisulfite)wefilteredoutreadsinwhich3ormoreCs
documentedmultiplegeneswhosemRNAsaresubjectto were not converted into Ts, to avoid assignment of m5C
RNA modification, suggesting that similar to eukaryotes, modification due to experimental artifacts. Although several
these organisms may utilize mRNA modifications as a modifications other than m5C may result in the absence of C-
mechanism forgeneregulation. .U conversion following bisulfite treatment, we designated all
non-converted sites as putative m5C sites, for the sake of
usage for RNA m5C methylation interrogation has been limited consistency (seeDiscussion).
untilrecently.Asaresult,nostudyhasaddressedthepresenceof WebeganbyexaminingmethylationsitesintRNAsandrRNAs
m5C sites, or any other RNA modification, in prokaryotes in a that passed a statistical metric (p,0.01) and in which the
transcriptome-wide manner, and no modification to bacterial or methylation levels exceeded 50%. Analysis of the data yielded a
archaeal mRNAhas beendescribed todate. totalof10sitesinrRNAsand85sitesintRNAs.Belowweprovide
Here, we set out to generate transcriptome wide maps of m5C asummaryoftheidentifiedmethylatedsitesinthedifferentRNA
RNAmodificationsitesinrepresentativemodelorganismsfromall classes ineach oftheorganisms.
threedomainsoflife.WeappliedbisulfitetreatmentontotalRNA
fromtheseorganisms,andgeneratedacomputationalpipelinethat Methylation of rRNA molecules
identifiesmethylatedpositionsafteraccountingforvariousartifacts. In E. coli we identified rRNA modifications at 3 distinct
Thisapproachwashighlysensitive,andallowedtheidentification positions.Theidentifiedresiduesincludedposition967inthe16S
andquantificationofthevastmajorityofknownm5Cpositionsin rRNA subunit, and position 1962 in the 23S rRNA subunit
tRNAsand rRNAsintheseorganisms, aswellasthedetection of (Figure 2A–B). Both these residues have been previously deter-
novel positions in these structural RNAs. Anti-m5C RNA immu- minedtocontainm5Cmodifications[25–27].Inaddition,position
noprecipitation confirmed our findings. Intriguingly, we detected 1402 in the 16S rRNA of E. coli was identified as having a
m5CsitesinseveralmRNAsinarchaea,andidentifiedasequence methylationlevelof58%.ThispositionisknowntoharboraN4,2-
motifguidingtheseRNAmethylations.Thismightbesuggestiveof O-dimethylcytidine modification which also is not amenable to
an additional layer of regulatory complexity present – potentially bisulfiteconversion[21],emphasizingthepossibilitythatafraction
havingfunctionalconsequence–onprokaryoticmRNAs. ofthesiteswedetectedmayreflectmodificationsotherthanm5C
(see Discussion).
Results We identified twomodified residuesat positions 977and1411
of the 16S rRNA of B. subtilis (Figure 2C). While neither one of
To obtain a single-nucleotide resolution overview of m5C sites these residues has been previously directly characterized as
present in RNA, we extracted total RNA from four model methylated in B. subtilis, they are positionally orthologous to the
organisms, spanning all three domains of life: the eukaryote previouslycharacterizedpositions967and1402intheE.coli16S
Saccharomyces cerevisiae, the archaeon Sulfolobus solfataricus, and the rRNA,respectively(above).Notonlywerethepositionsconserved,
grampositiveandnegativebacteria,BacillussubtilisandEscherichia but also the methylation stoichiometry appeared to be conserved
coli, respectively. We treated the RNA with bisulfite using a betweenE.coliandB.subtilis,withmethylationlevelsof98.7%at
modified version of the protocol for bisulfite-treatment of position977comparedto97.5%inE.coli,and63.7%atposition
DNA,(described in [21]), prepared a pooled cDNA library from 1411compared to58%inE. coli.
the four organisms, and subjected them to massively parallel In S. solfataricus we identified one m5C site at position 1369 of
sequencing(Table1;Figure1A).Sinceallmodifiedsitesidentified the16SrRNA,andanotheratposition2643ofthe23SrRNA.An
to date have originated from either tRNAs or rRNAs, we used a additional site with a methylation level of 43% was observed at
protocolthatenrichesfortRNAandrRNAmoleculesduringthis position2121ofthe23SrRNA(Figure2E–F).Position1369atthe
initial approach. This allowed us to test the specificity and 16S was previously characterized as subjected to an unknown
sensitivity of the assay (Methods). Sequencing reads from this modification[28],whereasnoevidenceexistedformethylationof
experimentwereobtainedprimarilyfromtRNAandrRNA,thus the23Ssites.Sangersequencingconfirmedthatthesesitesdidnot
validating the approach. Sequencing results gave sufficient undergo bisulfite conversion, providing further support for their
PLOSGenetics | www.plosgenetics.org 2 June2013 | Volume 9 | Issue 6 | e1003602
m5CRNAModificationinthe3DomainsofLife
methylation(Figure3).Thepresenceofthesemodificationsiswell ed
in line with previous chromatography/mass-spectrometry based ver
analysis, which predicted one m5C site in the 16S subunit of S. co
s
solfataricus, and1–2additional sites inthe23Ssubunit [29]. A
N
In yeast we identified two modified positions within the 26S tR % % % %
% 4 6 9 9
rRNA subunit. Position 2278, which was found to have a 7 9 8 6
methylation level of 72%, was previously reported to contain a
methylatedsite[30].Toourknowledge,wearethefirsttoreport
the presence of a methylation site at position 2870 (methylation with.5
e
lteiovnelo9f8t%hi,sFdiegtuercete2dDs)i.teIt(smp5rCesesintceeparnecdedtheednbuyclaeoptyidriemciodminpe)o,siis- eneserag
gv
consistentwiththepredictionofanadditionalm5Csiteinthe26S Aco
unit of yeastrRNA,whichwasnot mapped at thetime[30]. tRNean
#m 32 47 39 35
Methylation sites within tRNA molecules
Eukaryotic and archaeal tRNA molecules, but not bacterial
tRNAs, are known to undergo m5C modifications at specific A
N
positions. Consistent with this finding, we identified 46 modified R
t
sites in 34tRNAsinS. solfataricus, and39sites in30tRNAs inS. # 43 49 44 51
cdeirfefveirseianet. TtRoNaAllos,wwcoemgpeanreartaivteedanaalymsiusltoifplmeeathliyglnamtioenntleoveflsthine with.5
e
aarlocnhgaetahleamndbayseeadstotnRtNheAirs,manetdhycloaltoior-ncoledveedlsm(Feitghuyrlaete4d).pNoostiatibolnys, eneserag
gv
pinositthioisnsaenvaelynsiwshwenemexetahmylianteidonsliegvneilfsicwanetre(bp,elo0w.015)0%m,etthoyalalltoewd rRNAeanco
full comparative inspectionof thedata. #m 2 2 2 4
Consistent with previous findings, the methylated positions in
botharchaeaandyeastwereclusteredatpositions48–49alongthe
tRNA molecule (numbers based on E. coli tRNA positions, as in A
N
[31]). In archaea most (30/34, 88%) of the tRNAs were R
r
methylated at position 48,whileabout half of these (14/30)were # 2 2 2 4
alsomethylatedatposition49.Thepatternofm5Cmodificationin
yeast was different: usually, either position 48 or position 49 was
methylated, with only two tRNAs showing modifications at both as
positions. Two other tRNAs also contained modifications at ms. acil
position50in thevariable region. nis our
Therelativelyhighcoverageoftensandsometimeshundredsof ga nesdt
raaenbaldydsdcioifnfrerereeasnpchocentsdRiinNngAmtoeatlhtlhoyewlaeptdieornuceslnetvoteoqlfsutaaRtnNstiptAaecttiirvfaiecnlyspcoarssispitetisossnmssiom(pdirilfeaiesruidtmieas-t udiedor %cytosiconverte 98.3% 98.7% 97.6% 98.4%
those positions). In general, levels of methylation in yeast were st
e
significantly higher than those in archaea, with mean values of h
t
96% per position in S. cerevisiae as compared to mean values of n d
8T2h%ismobasyerimvepdlyinthaSt.tshoelfattyapriiccuasls(Mingalnen,-sWtrohnitgnlyeymteetsht,ylapt=ed3.s8itee-6in). NAsi appe ering.
ymneeeaetsdhtsylmtaotaebydecstoiatmkeespneinnwsiaStht.ecsoafloufarttiaotrnhic,eusa.sprToethsheiesnrciefnatceotrfoprrtsewtmoataioymnao,crechoouwwneetavkfeolryr, AsandtR #readsmtotRNA 42,425 168,557 5,626 18,782 apriortofilt
theobserved differences inmethylation levels (see Discussion). N at
R d
Surprisingly, a small fraction of the tRNAs were found to be r w
dperevsoeindceofofma5Ccytmosoindeifiicnaotinoensofinthethpeosviatiroianbsl4e8r–e5g0io.nT,hdeesespinitceluthdee ering ed thera
damLGttmaAtRRhnnaelleorttStcuNNuoetaidhhTec.ru,AAioovnCyfAgie--rdlaCchiaATdrnotat,taiCdolrntcibvaloipwTileoitAunsCaCilnauGo(dysdCpr,nliCddafAoniiia,tnosintsTgtleiatpieGt-hopniloacoCutSneudoshnr.caitnsteailtiudavtRots3yibnameonvp4pNlrtsdfeirees)ea2nAiettsto7mhgavot-nriyoRLoof5tio,lchfCffeauNumeyuttssRtReA.elsTRraydiaoNAN-tyNseATlAteApAsirA,rhdoeog-wtiebspsPmfsRCineulrttCamrNitohotoisceTdfieeAnigaisgieloinsfem-hvdmibiLcdcnetwstoa5eneipCdtvemuprioreyiovtrfeehseeoiini[tcnaedtvhs3aoaiis,io2yrbtttbdihinln]snsoear,eeeoneat5nsSriltnea6oav.acodsnetedsntoidpoRveflwtoeftNtotiRaeitnlRRenfytecAaNlntNNlrilosittAtboc.AAhhiuaualwseeers-s-.l SequencingreadscovTable1. #readsmappOrganismtorRNA B.subtilis4,786,857 E.coli206,162 S.solfataricus12,654,256 S.cerevisiae1,215,559 aConversionrateswerecalculatedoutofdoi:10.1371/journal.pgen.1003602.t001
PLOSGenetics | www.plosgenetics.org 3 June2013 | Volume 9 | Issue 6 | e1003602
m5CRNAModificationinthe3DomainsofLife
Figure1.Flowofthebisulfite-treatment/RNA-seqexperiment.(A)ShownisaschematicrepresentationofRNAmolecules;coloredboxes
representsinglebases.Red,blue,greenandblackrepresentC,U/T,A,andGresidues,respectively.m5Cmodifiedcytosinebasesaremarkedbya
blackcircle.TotalRNAisbisulfite-treated,leadingtodeaminationof‘C’residuesinto‘U’,exceptformethylatedresidues.Bisulfite-treatedRNAis
reverse transcribed into cDNA, which is then sequenced via an Illumina HiSeq system. This yields short sequence reads, representing random
fragments of the sequenced RNAs. Resulting reads are mapped to the genome using an algorithm that allows mapping of ‘T’ residues in the
sequencedcDNAsontogenomic‘C’s.Residuesinwhich‘C’sarefoundtobeconsistentlynon-modifiedaredeclaredasm5C(redarrow).(B)Flowchart
ofdataanalysisandartifactfiltering(Methods).
doi:10.1371/journal.pgen.1003602.g001
methylationlevels,thesesitesrequirefurtherverification.Finally, deplete tRNAs, combined with rRNA depletion protocols to
we failed to detect the known methylation at position 40 of enrich for mRNAs (Methods). For S. cerevisiae we made use of a
tRNA-Phe[6]duetolackofsequencecoverageforthisparticular dataset of short sequences previously obtained using bisulfite-
tRNA. treatment of polyA-selected RNA [33].
We used the computational pipeline outlined above toanalyze
Methylation in mRNA molecules the libraries obtained from the mRNA enrichment protocols.
Wewerenextinterestedinsearchingformethylatedsiteswithin Coverage along mRNA in this experiment was substantially
mRNA molecules. Since our initial experiment lacked sufficient improved, with sufficient coverage (mean coverage $5 reads/
sequencecoveragetoidentifysuchsiteswithinthevastmajorityof nucleotide)in1,394proteincodinggenesinB.subtilis,1,110genes
mRNAs due to the high coverage of rRNA and tRNAs, we in S. solfataricus, and 4,489 genes in yeast. In E. coli, due to very
performed a secondbisulfite-seq experimenton the bacterial and high levels of ribosomal RNA reads (.99%), only 287 protein-
the archaeal RNA using size-selected total RNA (.200bp) to codinggenes had sufficient coverage (Table2).
PLOSGenetics | www.plosgenetics.org 4 June2013 | Volume 9 | Issue 6 | e1003602
m5CRNAModificationinthe3DomainsofLife
Despite significant coverage of most yeast genes, we were only localizedtowardsthebeginningofthegene(Fig.S1,P=0.07).The
able to detect a single event of m5C on an mRNA in that apparentlynon-uniformdistributionofmodifiedsitesmayprovide
organism, occurring on a protein of unknown function. No m5C an angle for future elucidation of the function of this RNA
events were found in the expressed bacterial mRNAs that were modification.
coveredinourexperiment,althoughthelackofcoverageformost
E.coligenespreventsusfromdrawingastrongconclusionastothe Direct immunoprecipitation of methylated RNA
possibility of methylated sites inmRNAsfor that organism. Severalmodificationsotherthanm5Cmayresultinaninability
In S. solfataricus, the mRNAs of two protein coding genes to convert C-.U following bisulfite treatment, including 3-
contained a nucleotide methylated to a level of .50%, and 12 methylcytidine, N4-methylcytidine, N4,29-O-dimethylcytidine
additional genes contained nucleotides with methylation levels of and N4-acetylated variants [20,21]. To further examine the
20–50% (Table 3). Many of the genes harboring the m5C nature of the novel modifications we observed in S. solfataricus
modification were enzymes involved in energy and lipid metab- RNAs, we set out to conduct direct immunoprecipitation of
olism(notablyoxidoreductasesanddehydrogenases),whichmight modified RNA. For this, we used a monoclonal antibody that
imply a possible role for this modification in regulating specific specifically binds5-methylcytosine. Thisantibodyisbroadlyused
metabolic processes. Interestingly, all 14m5C positions in S. to specifically detect m5C modifications in DNA (e.g. [34]), but
solfataricusmRNAswerelocatedwithinastrongsequencemotifof sinceitwasraisedagainst5-methylcytosinenucleotideconjugated
AUCGANGU (Figure 5C). This same motif is found at the m5C toovalbuminwithouttheriboseordeoxyribosesugar,itisblindto
sites we recorded at positions 2121 and 2643 of the S. solfataricus the DNA/RNA context of the modification and hence binds the
23S rRNA (Figure 2). These results suggest that the same RNAformofm5Caswell.Asacontrolweusedasecondantibody
machinery (probably a methyltransferase) is responsible for m5C that specifically binds 5-hydroxymethylcytosine (hm5C) but not
modifications both on the 23S rRNA and on the mRNAs. m5C. The hm5C modification is similar to m5C but carries an
Moreover, our results imply that this machinery recognizes a additional hydroxyl group on top of the added methyl group.
consensus sequence of AUCGANGU. The identification of a Total RNA of S. solfataricus was sheared into ,100nt-long
strong consensus motif reinforces the validity of our bisulfite- fragments (the ‘‘input’’) and immunoprecipitated using the anti-
sequencing approach in identifying real modified sites on RNA m5C or anti-hm5C antibodies. Libraries were prepared from
bases, and supports the existence of a single predominant immunoprecipitated as well as non-immunoprecipitated input
methylation machinery on mRNAsin S.solfataricus. control RNA fragments, and subjected to massively parallel
Toverifythatthem5Cmodificationswedetectedreproducibly sequencing on theIlluminaplatform.
appear in S. solfataricus mRNAs we sequenced a second bisulfite- MappingtheresultingreadstotheS.solfataricusgenomeresulted
treatedbiologicalreplicatesampleoftotalRNAfromS.solfataricus. in a highly non-uniform coverage (Fig. 6). Peaks in the coverage
Indeed, all m5C sites identified in the first experiment were also wereclearlyobservedintherRNAattheexactlocationsidentified
detectedinthesecondexperimentwithsimilarmethylationlevels bythebisulfitesequencingasm5Cmodified(positions1369inthe
butwithhighercoverage(Table3).Toruleoutthepossibilitythat 16S and 2121 and 2643 in the 23S). These peaks represented
the consensus motif we identified is intrinsically resistant to enrichmentof18–30foldovertherestoftherRNAmolecule,but
bisulfite conversion, we mixed the second S. solfataricus RNA no enrichment was observed in these positions in the non-
sample, prior to bisulfite conversion, with two synthesized 200nt immunoprecipitated(input)sampleorthesampleimmunoprecip-
RNA fragments each harboring a common representation of the itated by the hm5C antibody. Most (10/14, 71%) of the sites we
consensus motif (either ATCGAGGT or ATCGAAGG; Meth- identifiedinthemRNAsofS.solfataricus(Table3)werealsofound
ods). Despite very deep coverage obtained for the two artificial to be enriched in the immunoprecipitated library (Fig. 6C–E,
consensus-bearing RNAs (130,000 and 2.8 million reads, respec- enrichment of 10–35 fold). These results provide strong indepen-
tively),noevidenceform5CwasobservedwithintheseRNAs,and dent verification that the sites we identified in the rRNAs and
the percentage of cytosines that were not converted into uracils mRNAsofS.solfataricusindeedcorrespondtom5Cmodifications.
within the two synthesized consensus motifs was 0.6%, similar to
non-methylated residues (Table 2). These results show that the
Discussion
methylation sites we observed in S. solfataricus mRNAs do not
represent a motif-dependent artifact. WehaveemployedbisulfitetreatmentofRNA,combinedwith
To determine if the presence of this consensus sequence is high-throughput sequencing, to generate a sensitive map of
sufficienttodrivemethylation,wesearchedforthemotifwithinall methyl-modified cytosines in four model organisms across the
S.solfataricusproteincodinggenes.Weidentified75sitesthatwere microbialtreeoflife.Ourmapnotonlyrecoversthevastmajority
coveredbyatleast 5reads(regardless ofp-valuescore).Ofthese, of known sites within tRNAs and rRNAs in these organisms, but
31 (41%) were methylated at levels .10%, and 13 (17%) were also provides a measurement of methylation efficiency for each
methylatedatlevelshigherthan20%.Theselevelsarefarhigher position, andreveals novelsites withinrRNAsandmRNAs.
than expected by chance alone: examining all sites (n=22,665) Several modifications other than m5C may result in a lack of
covered by at least 5 reads, but significantly differing from the CRU conversion following bisulfite treatment [20,21]. Indeed,
consensus sequence, only 5.9% had methylation levels of .10%, and in agreement with previously published data [21], position
and 0.9% of the sites had methylation levels exceeding 20%. 1402inE.coliidentifiedinthisstudyisknowntoharboranN4,29-
These results suggest that the presence of this motif is to a large O-dimethylcytidinemodification,whichisknowntoberesistantto
extent sufficient for recognition and methylation by the putative bisulfite conversion. Therefore, the novel modified positions we
methyltransferase. However, the varying levels of methylation reportinthisstudymightcorrespondtomodificationsotherthan
suggestthatadditionalfactorsotherthantheimmediatesequence m5C. To verify that the novel modifications in mRNAs we
environment –suchasRNA availabilityormoredistant motifs– identifiedinS.solfataricusindeedrepresentm5C,weutilizedm5C-
mayalso playa role indetermining methylation levels. specific antibodies that are usually used in pulldown experiments
We next examined the relative position of the modified sites ofmodifiedDNAmoleculesthatareanepigeneticmarkerforgene
within the genes they reside. Methylated bases tended to be silencing in mammals. Since these antibodies also bind the RNA
PLOSGenetics | www.plosgenetics.org 5 June2013 | Volume 9 | Issue 6 | e1003602
m5CRNAModificationinthe3DomainsofLife
Figure2.Sitesofm5CidentifiedinrRNAmoleculesofthestudiedorganisms.(A–F)Eachpanelpresentsasequencewindowarounda
detectedmethylatedsite.X-axis,positionalongtherRNAmolecule;Y-axis,numberofsupportingreadsperposition.Positionshighlightedinyellow
arethemethylatedones,withthemethylationratioindicatedbythefractionofresiduesinwhicha‘‘C’’residuewasnotconvertedinto‘‘U’’.
doi:10.1371/journal.pgen.1003602.g002
formofm5C,theyformedanidealtoolforindependentvalidation elucidate the possible function of these modifications. In partic-
of our bisulfite-based findings. Each of the approaches has its ular, profiling of the methylation status across different cellular
strengthsandweaknesses:whilethebisulfiteconversionprovidesa states,orinresponsetoexternalstimuli,willallowinvestigationof
single-nucleotideresolutionpositioningofthemodifiedsite,itmay the extent to which this modification is dynamically regulated.
also report other types of modifications. The antibody-based Knockoutstudiescanprovidebetterunderstandingoftheenzymes
approach provides specificity to a single type of modification but involvedinmediatingthismodificationacrossdifferentorganisms
with lower resolution of about 100bp [35]. Therefore, the andwillbe crucialforunderstanding its function.
combination of bisulfite-conversion and RNA-immunoprecipita- Our identification of a defined sequence motif associated with
tion provides synergisticresults. all mRNA methylated positions in S. solfataricus strongly suggests
Ineukaryotes,thechemicalmodificationofmRNAsisemerging thatthesemodificationsindeedoccurinvivoandarenotanartifact
asanimportantfactorintheregulationofgeneexpression.These of the detection method. This motif, along with the recently
modifications have been shown to affect mRNA translation, reported sequence motif associated with m6A modifications on
splicing,stabilityandtransport[20,35,36].Toourknowledge,our mammalian mRNAs [35], are the only reported cases of clearly
report presents the first evidence for mRNA modification in defined linear sequence motifs directing multiple RNA methyla-
archaea,openingawindowintoapossibleadditionallayerofgene tionevents.Indeed,studiesonvarioustRNAandrRNAmodifying
regulation in archaea. Further studies are needed in order to enzymeshadsuggestedpreferenceforlocalRNAstructuresrather
PLOSGenetics | www.plosgenetics.org 6 June2013 | Volume 9 | Issue 6 | e1003602
m5CRNAModificationinthe3DomainsofLife
Figure 3. Sanger-based verification of two novel methylated positions inS.solfataricusrRNA identified using RNA-seq. Bisulfite-
convertedsequenceswereamplifiedusingamplicon-specificprimersandsequenced.(A)PositionC1369intheS.solfataricus16SrRNA.(B)Position
C2643intheS.solfataricus23SrRNA.
doi:10.1371/journal.pgen.1003602.g003
thanforaspecificsequence[25,37,38].Inthisrespect,itispossible rRNAs have been extensively characterized, it is highly probable
that there is a fundamental difference between tRNA and rRNA that the majority of these sites represent artifacts from various
molecules, in which modifications are often sequentially added sources. This notwithstanding, this group contains a single site in
according to folding and maturation levels, therefore demanding the E. coli 16S rRNA (position 1407), which is, indeed, known to
structuraldependency,andmRNAs,inwhichthelinearsequence be modified. This suggests that there may be additional real
maybe themostimportant determinant. methylatednucleotideswithinthisgroup.However,ourapproach
The motif we identified in S. solfataricus, AUCGANGU, is also is currently unable to differentiate between rarely methylated
found at the modified positions C2121 and C2643 in the 23S positions andexperimental artifacts.
rRNA. Therefore, it is likely that the same methyltransferase Theemergenceofultra-highthroughputsequenceinterrogation
responsible for modification of these rRNA residues also methyl- technologies is revolutionizing research on RNA modifications.
ates the positions we detected in mRNAs. It is possible that the Applicationofsuchtechnologies,includingRNA-seqandRIP-seq,
modificationswefindonmRNAsmightreflecta‘‘spillover’’ofthis to study such modifications in eukaryotes has recently revealed
enzymes’ activity onto non-specific mRNA substrates. However, thatmRNAscarryaplethoraofconservedmodifications,suchas
nomethylatedsiteonS.solfataricusmRNAsresemblesthemodified A-to-I [40], m6A [35] and m5C [20]. Nevertheless, the field of
sequenceofthe16SrRNAinthatorganism,suggestingaspecific mRNAmodificationsisstillinitsinfancy,andevenineukaryotes,
activity for the23S methylase, but not forthe 16S methylase, on where several modifications have already been extensively
mRNA positions. Interestingly, recent work by Squires and characterized, their functional consequences are still poorly
coworkers similarly found that m5C modifications on human understood. Our report that mRNAs of archaea also carry such
mRNAs are mediated by the RNA methyltransferase NSUN2, modifications raises the intriguing possibility that mRNA modi-
previously knowntoactonly onhuman tRNAs [39]. ficationsinprokaryotesformanadditionallayerofgeneregulation
Our approach not only detects methylated positions in RNAs, that has not yet been addressed. Whether them5C modifications
but also provides a quantitative measure of the fraction of are functionally relevant, and whether other RNA modifications
transcripts harboring this methylation (to which we refer as also existonprokaryotic mRNAs, remains tobedetermined.
‘‘methylation level’’). The close similarities in rRNA methylation
levelsoforthologouspositionsbetweenthegrampositivebacterium Methods
B.subtilisandthegramnegativeE.colireinforcesthevalidityofthis
quantitative measure. Nevertheless, this measure should be taken RNA samples preparation
with caution, as factors such as miniscule DNA contamination Escherichiacoli(MG1655)cellsweregrowninLBmediumtolog
(which is non-methylated), for example, could contribute noise to phase at 37uC. Bacillus subtilis str. 168 cells were grown in LB
this measurement. In addition, bulky modifications (which are mediumtomidlogphaseat37uC.S.solfataricusP2(DSMZ1617)
prevalentintRNAs)mayhinderreversetranscriptaseprocessivity, cellsweregrownindefinedmodifiedBrock’smineralmediumwith
and hence, completely modified tRNA molecules may be under- final pH 3.5, and S. cerevisiae (BY4741) cells were grown in YPD
represented in the sequenced data. Therefore, the stoichiometric medium to log phase at 30uC. All cells pellets were suspended in
measurementswerecordedmightbebiased.Methodologiesthatare RNAlater (Ambion, AM7022) for 30min at room temperature.
not based on reverse transcription should be applied to decisively Pre-treatmentsforRNAisolationincludedLysozymedigestionfor
determinemethylationstoichiometry. E.colicells,glassbeadsvortexforB.subtilisandLyticasedigestion
An additional limitation of our approach is that it will fail to for S. cerevisiae. Total RNA was then extracted using Tri-Reagent
detect positions methylated at low levels, given the conservative (Molecular Research Center Inc.) according to manufacturer’s
cutoff we set of filtering out rarely methylated positions. Indeed, instructions.RNAsamplesweretreatedwithTurboDNA-freekit
apartfromthepositionswedescribedwithhighmethylationlevels, (Ambion) and rRNA was removed from the E.coli and B.subtilis
there were thousands of additional positions with significant p samplesusingMICROBExpressmRNAenrichmentkit(Ambion).
values, but with much lower methylation levels. About 95% of
thesesiteshadmethylationlevelslowerthan10%,includingmany Bisulfite treatment
putativepositionsinrRNAsandtRNAs.Asmostofthesepositions Bisulfite conversion was performed based on the protocol by
havenotbeenpreviouslyreported,despitethefactthattRNAsand Schaeferetal.[21].Briefly,onemicrogramofeachRNAsample
PLOSGenetics | www.plosgenetics.org 7 June2013 | Volume 9 | Issue 6 | e1003602
m5CRNAModificationinthe3DomainsofLife
Figure4.Sitesofm5CidentifiedintRNAmoleculesofthestudiedorganisms.EachlinedenotesatRNAsequence,identified(left)byits
accessionnumberintRNAdb[41].Positionsalongtheconsensusareindicatedbelowthesequences,numbersbasedonE.colitRNApositionsasin
[31].TherelevantanticodonisgivenforeachtRNA.Methylatedpositionsarecolorcodedbytheirmethylationratio,accordingtothepresentedcolor
gradient(bottom).Forpositions48–50,numbersindicatetheproportionofreadssupportingmethylationoutoftotalreads(e.g.52/56indicated52
non-convertedreadsoutoftotalof56coveringreadsatthespecificposition).Top-block,tRNAsofS.solfataricus;bottomblock,tRNAsofS.cerevisiae.
doi:10.1371/journal.pgen.1003602.g004
PLOSGenetics | www.plosgenetics.org 8 June2013 | Volume 9 | Issue 6 | e1003602
m5CRNAModificationinthe3DomainsofLife
was incubated in DNA protect buffer and bisulfite mix, EpiTect
Bisulfite Kit (Qiagen), through 6 cycles of denaturation step at
s
ne 70uC,followedbyadeaminationreactionstepof1 hourat60uC.
e
g TheRNAwaspurifiedfromthebisulfitereactionmixusingMicro
g
nd Bio-Spin 6 columns (Bio-Rad) and treated with 0.5M Tris–HCl,
codivere 4% % 0% 0% pH 9 at 37uC for 1hour. RNA was then washed on YM-10
%co 33. 6.6 73. 72. microcon (Millipore) 5 times with 0.5ml ultra-pure water. RNA
samples were analyzed using the Bioanalyzer (Agilent) to assess
degradation status.
of
e
overagbgenes cDFNoArasllyn4tohregsainsisamnsd,uSnatrnegateerdsReNquAeanncdinbgisulfite-treatedRNA
cg were used as templates for cDNA synthesis using SuperScript II
nn
Meacodi 21.1 14.3 70.6 72.3 RerT’sp(Irnovtoitcroolg.ecnD)NanAdwraansdaommplihfieexdambyerPsCaRcc(oArdBignegneto)umsianngurfeagcutularr-
primers (for untreated RNAs) and primers specific to cytosine-
h
wit.5 dimeapmroivneatseedquseenqcuienngcreessu(flotsr,gbeinsuelrfiicteMtr1e3atseedquRenNcAess)(.coInntaoinrdinegratoll
ese
ng 4 bases) were combined with the 59 end of all the deaminated
ea
ger primer sequences. PCR products were separated on agarose gel
gv
#codinmeanco(exp.2) 1,394 287 c2,175 4,489 aosenfqdutheexnetcrePa.cCteRdupsrinogdugcetlewxtarsacctioonndkuictt(eQdiafgreonm).StahnegeMrs1e3qupernicminegr
Library construction and Illumina sequencing
h Equalamounts (50 ng)ofeach ofthefourorganism’s bisulfite-
wit.5 treated total RNA were mixed together. The mixed bisulfite-
ese treated RNA sample was used as a template for cDNA library
ng
geera preparation according to the mRNA-seq Illumina protocol,
#codingmeancov(exp.1) 1 41 744 1 oRDmNouiAtbtilnwegasstrtfhairensdtepfdroalygcAmD-eNbnaAtseedwdabsymgdReivNnaeAlreantpteducraiutfiisocinnatgsioaSntu9ps4etuerCpS.cfroiIprntI5Ibmrainiendf,.
s. random primers. cDNA was then end-repaired, adenylated and
m
end-ligated to adapters. Following gel separation, a ,200bp
s
ani d fragment was gel-purified. The cDNA library was further
org erte amplifiedandsequencedusing40single-readcyclesonaGenome
d nv Analyser II (Illumina).
e o
thestudi cytosinescauracils 9% 9% 8% 8% #eriment2. ATlei4cg0hn-nnmotloelgnointesgoSfrdernaedaBsdhswde,rhettapl:i/g/nwedwwu.sninovgocNraofvt.ocaolmign) to(Nboivsouclfritaef-t
sin %to 98. 98. 98. 95. exp converted genomes of Bacillus subtilis (NC_000964), Escherichia coli
gene nes ng..5in c(NereCvi_si0a0e0(9N1C3)_,0S0u1lf1ol3o3bu-Ns sCol0fa0t1ar1i4cu8s)(NdoCw_n0l0o2a7d5e4d) afrnodmSatchceharNomCyBceIs
eringproteincoding edto#codingge 4,179 4,376 2,979 6,235 therawdatapriortofilterigeneswithmeancoveragembined. wotetrawhRexsreeaseicNbgridegeiseAesnnin‘tdaes‘med–edl.dabaRlotne’po’3fned,p5aogaitnhdrtnnnhRgseedt,.tNtsrhfwoTuoeAacearomtshrdadeauolrilialdecilgttongcinenpwduirmoloraentrittedhii.rannveeePltegningaglwityrnioirnfaeniettrmcrosaemailmeitit‘nugith‘oem-nletrtnteihse6prddoeeu0l,effsegb-eamrhecydsenoen1fpotltoocw2hmierne0yosgeltsa-hebbitwneaqeasu4edsitnereht-ep-ndhlepoecer3aexsiag5riiinrteanis-dnlotgseoonftnr2shmmsogtee-tmileiFhnpyr,
SequencingreadscovTable2. #readsmappOrganismcodinggenes B.subtilis1,536,835 E.coli189,572 S.solfataricus1,216,554 S.cerevisiae5,161,198 aConversionrateswerecalculatedoutofbRelatestomeancoverageofallcodingcDatarelatestotwosequencedlanescodoi:10.1371/journal.pgen.1003602.t002 SrcfoItdabtmrhRdRonfieToloientduNtsNmDwlnhsteAariitAeecFsoiRtpetcuhsQ5nipnaNinleSnelrgoa,.Aot-ttafrnhRr1alosoider6c,gfNRnehSansuataAnrmtdnawnRadtdnsihdenaqbNdsnaostsueoctt[mA2cebr4po3aisma1pc,anr-Ss]otueured,oaprrld,amteRm6eiitneapx’ooeN’seddcl.netpisoAeaFsa,rfertngoeiwsgdwddaarvewoelieelnndtntrnehhscefuetrtuteosieercllaniiloatansnygsrselslgeiasiidtepgcnnnoaeanromgoscofrspeclmahaecrdraiteeetieibropuasso,eosftrdanpeooavstgssdrumhfte:aeidc.eotisetFuhnhTnRef-ieseonbrhtNasutaotesstaorsfAa,atermiiodtstadnvohriteeodfgiesfcopbaaetotanurtrntRtfsaaeeRdaieebNinsvlysnaNmeacfAeconseooAshdersstff.
PLOSGenetics | www.plosgenetics.org 9 June2013 | Volume 9 | Issue 6 | e1003602
m5CRNAModificationinthe3DomainsofLife
Genedescription transposaseISC1217 oxidoreductase transcriptionalregulatorLrs14 oligo/dipeptidetransport,ATPbindingprotein quinoloxidase-2,subunitI/III,cytochromeaa3 hypotheticalprotein heterodisulfitereductase,subunitB acyl-CoAdehydrogenase pyruvic-ferredoxinoxidoreductasedeltachain 30SribosomalproteinS2 CRISPR-associatedprotein,Csx7family transposaseISC1217 formatedehydrogenaseAlphasubunit enoylCoAhydratase proteinofunknownfunction
% p)
h. re % % % % % % % % % % % % % %
Met nd(2 53.4 45.2 35.8 23.8 27.8 32.8 34.5 15.7 14.9 19.7 10.5 10.7 15.1 35.4 NA
C
5
m
sg
dn
#ofreasupporti nd(2rep) 23 52 179 103 169 21 144 65 95 35 22 15 19 89 NA
#readscoveringposition nd(2rep) 43 115 500 432 607 66 417 414 637 178 208 146 126 251 NA
% p)
h. re % % % % % % % % % % % % % % %
Met (1st 70.0 56.7 47.7 37.3 36.7 33.3 30.0 29.4 27.3 27.1 26.7 24.0 22.2 21.7 92.3
C
5
m
sg
dn
reaorti ep)
#ofsupp st(1r 7 17 31 22 22 10 21 25 24 16 8 12 8 10 12
#readscoveringposition st(1rep) 10 30 65 59 60 30 70 85 88 59 30 50 36 46 13
Pvalue 0.003096 6.19E-07 8.06E-12 4.27E-08 4.48E-08 0.000797 1.63E-07 7.51E-09 1.94E-08 9.42E-06 0.004575 0.000231 0.005056 0.001131 2.69E-06
Str + + + + + 2 + + + 2 + 2 2 2 +
n
o
siti
mRNAs. 5mCpo 1501851 2808514 958127 1101881 2718024 578303 971413 2280456 1955967 55904 1278822 839015 2585238 1141274 619291
n
i
s 3
e 0
5dmCbas Locustag SSO1649 SSO3054 SSO1108 SSO1276 SSO2973 SSO0673 SSO1129 SSO2511 SSO2128 SSO0067 SSO1425 SSO0980 SSO2824 SSO1311 YMR178W en.1003602.t0
e g
difi al.p
Mo urn
Table3. Organism S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.solfataricus S.cerevisiae doi:10.1371/jo
PLOSGenetics | www.plosgenetics.org 10 June2013 | Volume 9 | Issue 6 | e1003602