The Evolution of LINE-1 in Vertebrates

23 Pages·2017·2.67 MB·English

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The Evolution of LINE-1 in Vertebrates
Ste´phane Boissinot1,* and Akash Sookdeo2
1NYUAbuDhabi,SaadiyatIslandcampus,AbuDhabi,UnitedArabEmirates
2DepartmentofBiology,QueensCollege,CUNY
*Correspondingauthor:E-mail:[email protected].
Accepted:October 3, 2016
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Abstract loa
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TheabundanceanddiversityoftheLINE-1(L1)retrotransposondiffergreatlyamongvertebrates.Mammaliangenomescontain fro
hundredsofthousandsL1sthathaveaccumulatedsincetheoriginofmammals.Asinglegroupofverysimilarelementsisactiveata m
h
timeinmammals,thusasinglelineageofactivefamilieshasevolvedinthisgroup.Incontrast,non-mammaliangenomes(fish, ttp
amphibians,reptiles)harboralargediversityofconcurrentlytransposingfamilies,whichareallrepresentedbyverysmallnumberof s://a
recentlyinsertedcopies.WhythepatternofdiversityandabundanceofL1issodifferentamongvertebratesremainsunknown.To c
a
addressthisissue,weperformedadetailedanalysisoftheevolutionofactiveL1in14mammalsandin3non-mammalianvertebrate de
m
modelspecies.Weexaminedtheevolutionofbasecompositionandcodonbias,thegeneralstructure,andtheevolutionofthe ic
differentdomainsofL1(50UTR,ORF1,ORF2,30UTR).L1sdiffersubstantiallyinlength,basecomposition,andstructureamong .ou
p
vertebrates.Themostvariationisfoundinthe50UTR,whichislongerinamniotes,andintheORF1,whichtendtoevolvefasterin .c
o
mammals.ThehighlydivergentL1familiesoflizard,frog,andfishsharespecies-specificfeaturessuggestingthattheyaresubjectedto m
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thesamefunctionalconstraintsimposedbytheirhost.Therelativeconservationofthe50UTRandORF1innon-mammalianverte- b
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barrmatsesrascueg,gaessitssothbasetrtvheedreinprmesasmiomnaolfs.transpositionbythehostdoesnotactinasequence-specificmanneranddidnotresultinan /article
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Key words: LINE-1, L1, vertebrate, molecular evolution. b
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Introduction 2
Anotherdifferencebetweenmammalsandnon-mammals /3
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TheLINE-1(orL1)non-LTRretrotransposonisoneofthemost resideinthemodeofevolutionofL1(fig.1).Inmammals,only 8
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widely distributed transposable elements in vertebrate ge- the most recently evolved group of elements is active at a /26
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nomes(TollisandBoissinot2012).Theabundanceanddiver- giventimesothatasinglefamilyofprogenitorisusuallypro- 0
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sity of L1 differs considerably among vertebrates, and is ducingnovelinsertions.Inthelong-term,thismodeofevolu- 41
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probably one of the genomic features that show the most tionresultsinaladder-shapedphylogeny,demonstratingthe y
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variationinthisgroup.Atoneendofthespectrum,mamma- replacement of one family by a younger one, and so forth u
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liangenomeshostanextremelylargenumberofL1insertions (Smit et al. 1995; Furano 2000). This mode of evolution is t o
thathaveaccumulatedsincetheoriginofmammalsandac- consistent with an arms race between the host, which n 1
0
count for close to 20% of their mass (Lander et al. 2001; represses L1 transposition, and L1, which evolves to bypass A
p
Mnoonu-mseaGmemnaolmianevSeerqteubernactiensgareetraelp.r2e0se0n2t)e.dInbycomnutrcahsts,mLa1lleinr rheigphrelyssdioivnebrgyetnhtefhaomsitl.ieCsoanrveecrsoenlyc,uirnrernetplytileasctaivnedinfisthh,esesvaemrael ril 20
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copynumbers,fromafewhundredstoseveralthousandel- genome. These active families have coexisted for extended 9
ements, representing <0.5% of their genome size (Hellsten period of time, since their divergence may pre-date the
etal.2010;Howeetal.2013).Thisislikelyduetoahigherrate originofvertebrates(Furanoetal.2004;Novicketal.2009).
ofDNAdeletioninthesegenomesbutcouldalsoreflectvar- ThedifferencesintheevolutionarydynamicsofL1among
iations in the rate of fixation of novel insertions, or both vertebrateshavefar-reachingconsequencesbecauseL1activ-
(Duvernell et al. 2004; Furano et al. 2004; Novick et al. ity has considerably influenced other genomic features and
2009;Blassetal.2012;TollisandBoissinot2013). sinceL1insertionscanbebothasourceofdeleteriousalleles
(cid:2)TheAuthor2016.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/by-nc/4.0/),whichpermits
non-commercialre-use,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Forcommercialre-use,[email protected]
GenomeBiol.Evol.8(12):3485–3507. doi:10.1093/gbe/evw247 AdvanceAccesspublicationOctober19,2016 3485
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FIG.1.—PatternofevolutionofL1familiesinmammalsandnon-mammals.ThephylogeniesareMLtreesbasedonthedataofKhanetal.(2006)and ic
Novicketal.(2009).Thenumberofcopyforeachfamilyisindicatedinbold.(A)ThisphylogenyrepresentstheevolutionofL1familiesinhumanand .ou
p
demonstratestheladder-likemodeofevolutiontypicalofmammals.(B)ThisphylogenyisbasedonlizardL1families(Novicketal.2009)andistypicalofnon- .c
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mammalianvertebrates(reptiles,amphibiansandfish). m
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(Boissinotetal.2006)andevolutionarynovelties(Warrenetal. drives transcription of the full-length L1 transcript (Swergold rtic
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2015).Itisthusimportanttodeterminethemechanismsre- 1990; Severynse et al. 1992; DeBerardinis and Kazazian -a
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sponsibleforthesedifferences.Anumberofstudieshaveex- 1999). The 50UTR of the mouse and rat L1 is bipartite and stra
amined the population dynamics of L1 insertions in consistoftandemarraysofmonomers(~200bpformouse, c
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mammalian (Boissinot et al. 2006; Witherspoon et al. 2006; ~650bpforrat),whichcontainCpG-islandandtranscriptional /1
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Rishishwar et al. 2015) and non-mammalian species signals,connectedtoORF1byan~250-bpregioncalledthe /3
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(Duvernell,etal.2004;Blassetal.2012;TollisandBoissinot tether (fig. 2A) (Adey, Tollefsbol, et al. 1994; Furano 2000). 8
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2013)butnostudieshaveexaminedtheevolutionoftheL1 The 50UTR shows little or no homology among mammalian /26
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sequence across vertebrates. In fact, almost everything we speciesorevenamongfamilieswithinthesamespecies(Adey, 0
0
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knowaboutL1,fromitsstructuretoitsmechanismoftrans- Schichman, et al. 1994; Khan et al. 2006; Sookdeo et al. 1
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position, results from studies in mammals, with a focus on 2013). Evolutionary analyses in primates and rodents have y
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human and murine rodents (for a recent review, see demonstrated that L1 lineages have repeatedly acquired ue
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Richardsonetal.2015).Consideringthedifferenceintheevo- novel 50UTR, possibly in response to the host repression of t o
lutionary dynamics of L1 between mammals and non-mam- L1transcription(Jacobsetal.2014).Thehuman50UTRcon- n 1
0
malianvertebrates,itisunlikelythateverythingweknowfrom tains an anti-sense promoter on the negative strand (Speek A
p
mammalsappliestofishandreptiles.AnalyzingL1evolutionin 2001)andasmallORF,termedORF0,whichistranscribedand ril 2
a phylogenetically broader comparative context could cer- translatedbuthasnoknownfunction(Denlietal.2015). 0
1
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tainlyimproveourunderstandingofthebiologyofL1across ORF1 and ORF2 are both necessary for L1 transposition.
genomesbutalsogiveuspowerfulinsightsintomammalian ORF1 contains a coiled-coil domain (CCD), which promotes
L1biology. theformationofORF1ptrimers,anon-canonicalRNArecog-
L1 transpose through a process called target-primed re- nitionmotif(RRM)andahighlyconservedC-terminusdomain
verse transcription (TPRT) where reverse transcription of the (MartinandBushman2001;Martinetal.2003;Januszyketal.
L1 RNA into cDNA takes place at the site of insertion (Luan 2007; Khazina and Weichenrieder 2009). The function of
etal.1993;Costetal.2002).AtypicalmammalianL1element ORF1remainsobscurebutithasbeenshowntohavenucleic
is6–7kblongandcontainsa50UTR,twoopen-readingframes acid chaperone activity (Martin and Bushman 2001) and
(ORF1andORF2)anda30UTR(fig.2A).L1insertionstypically recentstudiesshowedthatthehumanORF1prequiresphos-
end with an A-rich tail and are flanked by short (<10 bp) phorylationforretrotranspositioninacellculture-basedassay
targetsiteduplication.InmodernhumanL1,the50UTRcon- (Cooketal.2015).ORF1pparticipateintheformationofL1
tains a CpG island and acts as an internal promoter, which ribonucleoproteinparticles(RNP),whichisanecessarystepof
3486 GenomeBiol.Evol.8(12):3485–3507. doi:10.1093/gbe/evw247 AdvanceAccesspublicationOctober19,2016
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FIG.2.—(A)Typicalstructureofhumanandmurinerodentsfull-lengthL1elements(CCD=Coiled-coildomain;RRM=RNArecognitionmotif;CTD=C- /26
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terminaldomain;EN=Endonucleasedomain;RT=Reversetranscriptasedomain).(B)Schematicstructureoffull-lengthL1familiesinmammals,lizard,frog 0
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andzebrafish. 41
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the transposition process (Kolosha and Martin 1997; Kulpa 30 endofORF1overlapswiththe50 endofORF2.Thedicis- st o
andMoran2005).Interestingly,recentstudiesshowedthat tronic structure of L1 is unusual in eukaryotes and it is still n
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interactionsofpurifiedORF1pwithnucleicacidsexemplified unclearhowtheORFsaretranslated.Twopossibilitieshave 0 A
ssetavbeirlaizlaotifotnheopfrtehdeictpeudtaptriovpeeTrtPieRsToifnttheermLe1dRiaNteP,(iCncalluladhinang beteeanl.2o0ff0e6re),do.nEeithfoerrethacehreOaRrFes,tworothrieborisboomsoemeenttrhyastitterasn(sL-i pril 2
0
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etal.2012).Thelatterpaperisparticularlyinformativeasit lated ORF1 scan through the IGR to ORF2 start codon and 9
demonstrated that rapid oligomerization between ORF1p reinitiatetranslation(Alischetal.2006).
trimers upon their binding to nucleic acid is essential for The ORFs of L1 are AT rich, with a strong A-bias on the
retrotransposition, a novel coiled-coil-dependent property positivestrand,whichcouldaccountforprematurepoly-ade-
which is conserved despite extensive remodeling of the nylationsignalsandinefficienttranscription,atleastincellcul-
coiled-coil during evolution. ORF2 is highly conserved ture based retrotransposition assays (Perepelitsa-Belancio and
among mammals and contains endonuclease and reverse Deininger2003;Hanetal.2004).The30UTRshowsverylittle
transcriptase domains (Mathias et al. 1991; Feng et al. conservationamongspecies,yetallmammalian30UTRscontain
1996). A short (~40 bp) inter-genic region (IGR) separates apoly-Gtractofunknownfunction(HowellandUsdin1997)
the two ORFs in human, whereas an IGR spanning several and end with a functional but weak poly-adenylation signal,
hundredbasepairswasfoundinmarsupials,megabatsand whichisoftenby-passedduringtranscription,resultinginthe
afrotheria(Yangetal.2014).MouseL1lacksanIGRandthe transductionof30 flankingsequences(Pickeraletal.2000).
GenomeBiol.Evol.8(12):3485–3507. doi:10.1093/gbe/evw247 AdvanceAccesspublicationOctober19,2016 3487
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BoissinotandSookdeo
Here,weperformedacomparativeanalysisontheevolu- 2016). COILS calculate the probability that a given protein
tionof L1 active families across mammalsand inthree non- sequence forms a coiled-coil structure. Protein domains that
mammalian vertebrates species. We demonstrate that the canformcoiledcoilstypicallyconsistofsevenresiduesrepeats
length,structure,andbasecompositionofL1differssubstan- (or heptads) with non-polar or hydrophobic residues at the
tially among vertebrates but is remarkably conserved within first (a) and fourth (d) positions of the heptads. The analysis
species, exemplifying the finely tuned co-evolution between was run with window width of 14, 21, and 28. Since the
L1anditshost.Weproposethatthesevariationsrevealfun- results were very similar among analyses, we present only
damental differences in the nature of the interactions be- the results obtained with the 28 residues window width. A
tweenL1anditsvertebratehosts. conservativecut-offof90%wasusedtodefineanaminoacid
as participating in a coiled coil structure. In our analysis, we
D
differentiated canonical heptads (a–b–c–d–e–f–g), non-ca- o
Materials and Methods w
nonicalcoiledcoils(regionswithahighprobabilitytopartici- nlo
Wecollectedfull-lengthcopiesfromthegenomeof14mam- pateintheformationofacoiledcoilbutwhichdeviatefrom ad
e
mrabalbsit(,thmeooupseoslesummu,r,ahrmumadainll,o,doegle,pghiaanntt,phaynrdaxa,,hraotr,sem,ocuoswe,, tfh–gecoarnao–nbic–acl–hge–pat–afd–gs)trauncdtunreo,nf-ocroeilxeadmcpoliel,reag–ibo–ncs–b(w–cit–hdl–oew– d from
andpig),anon-avianreptile(thegreenanoleAnoliscarolinen- probabilitytoparticipateintheformationofacoiledcoil). http
sis),anamphibian(theAfrican-clawedfrogXenopustropicalis), Aphylogenyofallactivefamilieswasbuiltusingthemaxi- s
andateleostfish(thezebrafishDaniorerio).Thecoordinatesof mumlikelihoodmethodandaLG+G+I+Fmodelofmutation, ://ac
a
the elements were retrieved from the repeatmasker tables asdeterminedbythemodelestimationtool,implementedin d
e
availableatthegenome.ucsc.eduwebsite.Eachelementwas MEGA5.0(Tamuraetal.2011).Therobustnessofthenodes mic
recoveredwith2kbofsequenceupstreamanddownstreamto wasdeterminedusing1,000bootstrapreplicates. .o
u
accuratelyidentifythestartandtheendofthefull-lengthele- Basecompositionandcodonusageweredeterminedusing p.c
o
ment.Onlyfull-lengthandrecentlyactiveelementswereused the CAIcal program at http://genomes.urv.es/CAIcal/ (last m
inthe analysisto limit the uncertaintiesinherent to the con- accessed October 20, 2016) (Puigbo et al. 2008). For each /gb
e
structionofconsensussequencesineachspecies.Aphyloge- codon, the Relative Synonymous Codon Usage (RSCU) was /a
neticanalysisusingORF2wasfirstperformedtoidentifyactive estimated (Sharp et al. 1986). The RSCU is defined as the rticle
orrecentlyactivefamilies,whichwererecognizedasmonophy- numberoftimeacodonisusedforagivenaminoaciddivided -a
b
leticclustersofelementswithbranchlength<2%divergence. bythenumberofsynonymouscodonsforthataminoacid.We stra
Toinsureaccuracy,weonlyusedfamiliesforwhichwecould alsocalculatedtwoestimatorsofcodonbias:Nc(Wright1990) ct/8
collectatleasteightfull-lengthgenomiccopies.Aconsensus andCAI(SharpandLi1987).Nc(e.g.,theeffectivenumberof /1
2
sequencewasthenderivedforeachactiveorrecentlyactive codonusedinagene)quantifieshowmuchtheuseofaspecific /3
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family. This approach was used for all organisms except for codondeviatesfromequaluseofallsynonymouscodonsfora 85
humanandmouse,forwhichweusedtheconsensidescribed given amino acid. Nc ranges in value from 20 (when each /26
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inKhanetal.(2006)andSookdeoetal.(2013). amino acid is exclusively encoded by a single synonymous 00
4
Sequences were manipulated and consensi were gener- codon)to61(whenallsynonymouscodonsareequallyrepre- 1
b
ated using Geneious 8.1.5, created by Biomatters and avail- sented). The parameter CAI (Codon Adaptation Index) esti- y g
ableatwww.geneious.com(lastaccessedOctober20,2016). matesthecodonbiasgiventhecodonusageoftheorganism ue
s
ThelocationoftheORFswasdeterminedusingtheORFfinder andtheGCcontentofthegene.Itrangesfrom0to1,1mean- t o
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toolimplementedinGeneious8.1.5andthepresenceoffunc- ingthatitisalwaysthemostcommonsynonymouscodonthat 1
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tional motifs was determined with the search tool at http:// is used and the codon bias is low. The codon usage of the A
p
www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (last accessed organisms was obtained from the codon usage database at ril 2
October 20, 2016). The level of identify among amino acid http://www.kazusa.or.jp/codon/ (last accessed October 20, 0
1
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sequenceswascalculatedusingGeneious8.1.5.Searchesfor 2016). For mammals we performed the analysis with the
similarityamongregionsthatcouldnotbereliablyaligned(the humanandthemousecodonusageandweobtainedidentical
UTRs) were performed using DOTMATCHER at http://www. results.Sincethelizardcodonusagewaspoorlyrepresentedin
bioinformatics.nl/cgi-bin/emboss/dotmatcher (last accessed thedatabase,weestimatedthecodonusagefromthelizard
October 20, 2016). Repeated motifs were searched using cDNAentriesavailableinGenBank.Statisticalsignificanceof
the Tandem Repeats Finder website at https://tandem.bu. CAIisestimatedbycomparingtheobservedCAIvalueswith
edu/trf/trf.html (last accessed October 20, 2016) (Benson theexpectedCAI(oreCAI),whichdescribestherandomcodon
1999). The presence of known phosphorylation motifs was usageassumingtheGCcontentofthegenestudied.
determinedusingtheEukaryoticLinearMotifssearchengine RNA secondary structures were investigated using the
athttp://elm.eu.org/.ThepresenceandstructureofCCDswas RNAfold web server at the Vienna RNA web suite (Gruber
assessed using the COILS server at http://www.ch.embnet. et al. 2008). Putative Internal Ribosome Entry Sites (IRES)
org/software/COILS_form.html (last accessed October 20, were identified using the IRESPred tool, which uses 35
3488 GenomeBiol.Evol.8(12):3485–3507. doi:10.1093/gbe/evw247 AdvanceAccesspublicationOctober19,2016
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TheEvolutionofLINE-1inVertebrates
featuresbasedonsequenceandstructuralpropertiestopre- S2,SupplementaryMaterialonline).MammalianL1stendto
dictthepresenceofcellularorviralIRES(Kolekaretal.2016). belonger(7.1kbonaverage)thanthefrog(5.7kb),andfish
We also used the Viral IRES Prediction System (VIPS), which (5.8kb)L1s.Thereisnosignificantdifferenceinthelengthof
uses the secondary structure of four groups of known viral theelementsbelongingtothetwomainL1cladesinfrogand
IREStopredictputativeviralIRES(Hongetal.2013). fish.Inlizard,elementsofclade1aresimilartomammalianL1
inlength(6.4kbonaverage)butclade2elementsaresimilar
tofrogandfishwithregardtolength(5.4kb).
Results
SincethereisverylittlevariationinthelengthofORF1and
Wederivedfull-lengthconsensussequencesfor14mamma- ORF2,differencesamongvertebrateL1sarecausedbyvaria-
lianspecies,thegreenanole,theAfricanclawedfrogandthe tion in the length of the 50UTR, 30UTR, and IGR (table 2).
D
zebrafish.Asingleconsensuswasderivedforeachmamma- MammalianL1sandlizardclade1elementsarecharacterized ow
lian species because their genome hosts a single active (or by 50UTRs that are considerably longer (1.5 and 1.3 kb on nlo
recently active) L1 family, with the exception of the house average, respectively) than the fish, frog, and lizard clade 2 ad
e
mouse. The mouse genome contains three active families d
butonlyoneofthemwasincludedinthisanalysis(L1Md_A) L310UsT(0R.1ca6n,0a.l1so4,daifnfedr0g.2re3atklyb,armesopnegctfiavemlyil)i.esT,hyeeltetnhgetsheovfatrhiae- from
sincetheyaresimilarinsequenceandhavebeenanalyzedin h
tions do not follow a clear evolutionary pattern and the ac- ttp
dzeebtarailsfisehls,ewwehedreeri(vSeodo1k2d,e1o2e,taanl.d210713L1).Icnotnhseenasni,orlee,spfreocgt,ivaenlyd. Fqourisietixoanmopflelo,ntghe303U0UTRTsRsoeefmmsatmomhaavlsesohcocwursreadcsopnosriaddeircaabllyle. s://ac
a
ThesespeciesareknowntohostalargernumberofL1families rangeoflengthfrom148bpinhorseto994bpinelephant, de
(Furanoetal.2004;Novicketal.2009)butthestringencyof m
withanextremevalueof2,751bpinarmadillo.Similarlythe ic
thecriteriaweusedtoconstructfull-lengthconsensididnot 30UTRofthezebrafishrangesfrom167to807bp,withthe .ou
permitderivingconsensiforallactivefamilies.Thus,thedata- evolution of a very long 30UTR of 2,124 bp in the L1-11A p.c
o
setanalyzedhere consistsof 55consensussequences (avail- family. m
ableasfastafileinsupplementarymaterialS1,Supplementary ThepresenceandlengthofanIGRalsosignificantlyaffects /gbe
Materialonline). theoveralllengthoftheelements(table2,fig.2).Fish,frog, /artic
and lizard clade 2 elements have a relatively long IGR that le
PhylogeneticRelationshipsandDivergences ranges from 257 to 1,032 bp. Conversely, six out of the -ab
s
We first performed a phylogenetic analysis using the most nine lizardclade 1elementsare lacking anIGR, and for five tra
c
conserved region of L1, ORF2 (fig. 3). Mammalian L1 se- ofthose,ORF1andORF2overlap.MostmammalianL1havea t/8
quences form a monophyletic group with strong support. smallIGRrangingfrom26to82bp.Theexceptionsarethe /12
The lizard L1 elements also form a clade, composed of two opossum, elephant, hyrax, and pig, with IGR ranging from /34
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divergent sub-clades (Lizard clade 1 and clade 2 on fig. 3). 423 to 719 bp (fig. 4). Assuming that Afrotheria (elephant 5/2
Two highly divergent clades are also found in zebrafish and andhyrax)isthesistergrouptoallotherplacentalmammals 68
0
frog but these clades do not form species-specific groups, (Meredithetal.2011),wecaninferthattheancestralmam- 04
1
suggestingthattheirdivergencecouldhaveoccurredbefore malianL1hadalongIGRthatwaslostafterthesplitbetween b
y
thesplitbetweenteleosteanfishandtetrapods. AfrotheriaandtheotherplacentalsandthatthepigIGRwas g
u
TheidentityamongmammalianORF2rangesfrom48.4to acquiredindependently. es
76.1%(table1).Asexpectedtheidentitybetweentheopos- t on
1
sum L1 and the placental mammals is lower (48.4–53.6%) BaseContentandCodonUsage 0
A
than among placentals (58.2–76.1%). The identity between p
thetwomostdivergentcladesinlizard,frog,andzebrafishis To investigate the intrinsic constraints on LINE composition ril 2
and how this is influenced by the context of their host ge- 0
comparatively much lower, with average values of 26.5%, nomes, we compared GC content and codon usage across 19
27.5%,and31.2%,respectively.Theidentitywithineachof
species and among families within species. The overall base
thecladesisalsolowwithaveragevaluesof51.8%,38.5%,
content of L1 in vertebrates tends to be AT-rich, with GC
and 37.9% for clade 1 in lizard, frog, and fish, respectively.
contentrangingfrom33.9to48.1%.Thereareconsiderable
ThedivergencebetweenL1families,aswellasthephyloge-
differences in base composition among regions of L1 and
netic analysis, clearly indicates that each non-mammalian
among vertebrates (table 2), although the average genomic
genome contains a large diversity of L1 families, which is
GC content differs only moderately among vertebrates
veryancientandhaspersistedsincetheoriginofvertebrates.
(~41% on average in mammals, ~40.3% in lizard, ~40.0%
infrog,and~38.6%inzebrafish).Thereishoweververylittle
StructuralEvolutionofLINE-1inVertebrates
variationintheGCcontentofL1withinspecies,evenamong
Full-lengthL1elementsvarysubstantiallyinlengthamongand thedivergentlizard,frog,andfishfamilies(table2andsup-
withinorganisms(table2,fig.2andsupplementarymaterial plementarymaterialS2,SupplementaryMaterialonline).The
GenomeBiol.Evol.8(12):3485–3507. doi:10.1093/gbe/evw247 AdvanceAccesspublicationOctober19,2016 3489
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FIG.3.—MaximumlikelihoodphylogenyofL1familiesbasedonORF2aminoacidsequences.
3490 GenomeBiol.Evol.8(12):3485–3507. doi:10.1093/gbe/evw247 AdvanceAccesspublicationOctober19,2016
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LIZARD 1_AC20 22.1 21.5 19.3 19.3 20.4 23.2 21.5 19.3 17.6 34.1 31.8 OGL1-47 21.3 23.0 25.0 26.2 23.5 20.2 25.7 21.9 34.8 29.8 55.8 (contin
L R
M F
U
OPOSS 33.9 29.5 32.8 30.1 30.8 33.0 34.1 33.1 34.1 35.2 33.5 28.6 29.7 LIZARD 1_AC18 16.6 16.6 16.0 16.0 18.8 17.7 17.7 16.6 22.5 63.6 34.5 OGL1-46 24.3 28.7 25.3 22.7 24.3 23.5 26.2 26.5 33.0 35.8 42.5
L R
T F
N
A 0 8 6 4 4 9 6 5 8 8 0 7 2 9
ELEPH 50. 47. 45. 43. 51. 51. 43. 52. 55. 55. 53. 70. 50. LIZARD L1_AC17 21.5 21.0 17.1 17.1 21.0 19.3 19.9 17.7 23.1 43.5 34.1 ROGL1-3 24.3 26.5 28.1 19.9 23.2 25.1 24.0 24.3 31.3 32.8 34.5 Downloa
F d
X e
ARMADILLOHYRA 50.052.7 45.145.6 49.543.4 45.140.7 54.149.7 56.950.8 52.547.0 61.356.4 63.052.5 56.953.6 51.4 62.9 65.573.8 54.249.4 LIZARDLIZARD 1_AC14L1_AC15 48.926.7 47.825.0 53.425.6 52.825.0 50.026.7 67.426.1 76.426.7 27.8 37.5 26.827.1 25.425.5 26.026.5 OGL1-35FROGL1-38 55.624.9 65.027.1 52.524.7 53.128.2 52.226.0 56.024.6 54.427.3 27.1 28.0 27.832.1 29.334.0 28.033.4 d from https://academic
PIGCOW 53.855.5 50.053.8 51.148.4 47.846.2 54.754.1 57.553.6 54.747.5 64.661.3 63.0 74.5 65.666.7 61.962.6 64.864.9 53.252.8 LIZARD L1_AC_12L 50.0 49.4 56.2 55.6 52.8 67.4 79.2 38.1 27.2 25.8 26.5 FROGL1-32FR 51.1 54.4 50.3 51.1 50.0 44.0 40.6 27.3 27.9 26.6 27.8 .oup.com/gbe/a
HORSE 55.5 54.9 46.2 42.9 55.8 55.8 53.0 72.0 71.9 65.6 61.6 64.4 52.4 LIZARD 1_AC11 50.0 50.6 56.2 56.2 50.6 66.2 65.0 38.6 26.9 24.6 26.3 OGL1-29 46.7 55.5 52.5 44.5 42.9 32.7 34.7 28.0 25.0 27.0 24.8 rticle-abstra
A L FR ct/8
D 5 3 7 0 0 9 0 8 2 5 4 9 3 8 /1
fish GPAN 250. 147. 146. 144. 447. 61. 1 467. 267. 568. 763. 758. 861. 550. LIZARD L1_AC9 54.5 59.0 52.8 52.2 51.8 54.9 53.8 38.4 27.6 25.7 26.5 FROGL1-1 56.1 48.9 48.0 55.0 32.9 41.1 41.0 27.2 28.9 27.6 29.5 2/3485/26
Frog,andZebra RABBITDO 50.052. 51.645. 46.245. 46.740. 51. 66.9 64.776. 67.067. 68.368. 67.768. 64.763. 62.261. 63.863. 52.850. LIZARD L1_AC8 52.8 54.5 96.6 52.0 48.6 51.0 51.3 37.5 25.4 24.8 26.5 FROGL1-17 51.7 50.3 44.6 42.1 34.3 45.7 38.2 27.5 27.8 28.2 28.5 80041 by gues
mmals,Lizard, MOUSE 43.4 44.0 86.3 63.4 61.2 60.0 63.2 62.7 63.6 60.5 58.2 59.4 48.4 LIZARD L1_AC7 53.4 55.1 93.8 51.1 48.3 50.2 50.7 36.7 25.3 25.1 26.2 FROGL1-15 44.6 48.0 33.5 31.8 39.9 32.0 34.3 26.1 26.0 27.1 25.2 t on 10 April 2
FamiliesinMa EMURRAT 58.846.2 45.6 59.9 60.784.0 65.763.9 64.161.5 61.660.1 64.764.2 66.364.3 66.163.2 64.360.9 59.858.7 60.959.7 53.649.4 LIZARD L1_AC3b 90.4 53.2 53.1 49.8 49.6 50.3 50.5 37.4 28.1 26.8 28.3 FROGL1-11 53.3 32.9 40.7 42.5 35.0 40.2 50.4 29.7 27.2 27.1 28.7 019
1 L 6
L D 1 1-
among MAN 7.2 3.0 2.6 7.4 4.2 2.3 7.0 7.4 6.1 4.3 1.1 1.9 2.2 LIZAR L1_AC 82.7 52.4 52.7 50.0 48.7 49.8 50.2 38.2 28.0 27.5 28.2 FROGL 41.4 33.7 45.2 41.3 31.6 47.6 40.3 27.4 27.5 28.3 28.1
y U 6 6 6 6 6 6 6 6 6 6 6 6 5
ntit H b 1 12 4 5 7 8 0
e 1 3 7 8 9 1 _ 1 1 1 1 2
d C C C C C C C C C C C C
Table1AminoAcidI HUMAN LEMUR RAT MOUSE RABBIT DOG PANDA HORSE PIG COW ARMADILLO HYRAX ELEPHANT OPOSSUM LIZARDL1_A LIZARDL1_A LIZARDL1_A LIZARDL1_A LIZARDL1_A LIZARDL1_A LIZARDL1_A LIZARDL1_A LIZARDL1_A LIZARDL1_A LIZARDL1_A LIZARDL1_A FROGL1-6 FROGL1-11 FROGL1-15 FROGL1-17 FROGL1-18 FROGL1-29 FROGL1-32 FROGL1-35 FROGL1-38 FROGL1-39 FROGL1-46 FROGL1-47
FISH 1-17B 26.7 29.5 35.2 27.2 29.9 33.1 35.2 33.9 26.1 35.4 19.8 31.1 31.5 32.8 31.6 33.1
L
H 6B 6 7 7 2 9 7 7 0 8 1 5 6 7 2 7 4
FIS 1-1 30. 33. 39. 41. 29. 31. 33. 35. 27. 28. 17. 34. 33. 35. 33. 31.
L
FISH 1-13D 31.1 32.8 30.5 35.0 33.3 30.9 40.1 41.6 26.3 32.8 18.3 64.2 35.4 56.8 38.7 31.6
L
FISH 1-13C 32.0 37.0 36.2 37.8 36.2 33.0 40.1 42.5 31.8 32.2 19.5 61.1 35.8 54.2 38.8 29.9 Dow
L n
lo
a
FISH L1-13B 26.3 27.9 30.9 33.0 26.3 30.0 36.0 36.1 28.9 33.5 20.6 37.0 51.0 49.4 39.4 31.9 ded fro
m
h
FISH L1-13A 26.5 32.6 33.3 35.0 34.5 29.1 37.3 37.0 31.8 32.8 17.8 49.0 55.3 58.6 38.8 30.4 ttps://a
c
a
FISH 1-11Aa 20.7 16.6 19.5 20.2 17.1 20.2 21.2 20.0 16.8 19.8 35.6 35.1 34.1 34.2 36.3 32.7 demic
L .o
u
p
FISH L1-10B 29.5 32.4 29.9 26.1 40.7 34.5 29.4 30.5 26.8 34.2 34.7 36.3 35.7 34.7 35.7 31.7 .com/g
b
e
/a
FISH L1-8 26.4 27.5 25.1 29.3 30.2 21.8 30.7 31.8 32.2 33.5 31.4 32.0 32.2 31.9 34.6 32.2 rticle
-a
b
s
FISH L1-7C 29.2 32.0 32.8 36.1 30.9 27.4 68.2 32.3 35.3 35.0 44.4 46.9 46.0 45.7 40.7 31.3 tract/8
/1
2
/3
FISH L1-7B 27.3 30.7 33.9 34.1 33.9 29.4 60.1 32.4 36.1 35.5 46.3 48.1 47.8 45.4 40.6 32.3 485/2
6
8
0
0
FISH L1-6 29.3 27.1 28.8 23.8 33.7 33.3 31.6 32.8 29.9 33.8 27.8 30.9 30.0 30.6 32.0 42.6 41 b
y
g
u
FISH L1-1D 33.5 33.5 25.0 29.1 31.7 37.7 36.5 33.1 34.7 35.3 35.4 36.6 36.5 36.6 36.6 33.4 est on
.w 10
o A
FISH L1-1B 29.8 27.9 42.0 34.6 28.8 46.0 46.0 31.3 35.0 33.1 44.0 43.5 45.4 43.4 38.8 30.5 2bel pril 2
F 0
R 1
O 9
FISH L1-1 25.9 33.1 46.4 37.2 31.2 49.6 48.9 34.6 37.3 36.1 45.8 48.3 46.0 46.7 41.3 32.8 and
al
n
o
FISH 1-12B 47.5 33.6 31.5 34.2 29.1 31.7 30.8 30.0 30.2 30.6 30.7 30.5 30.4 30.9 31.9 28.8 diag
L e
v
o
b
FISH L1-12A 48.3 33.4 30.7 34.4 27.6 31.8 32.6 29.6 31.4 30.4 31.3 32.6 31.5 31.3 32.5 28.8 ORF1a
—
ed 12A 12B 1 1B 1D 6 7B 7C 8 10B 11Aa 13A 13B 13C 13D 16B 17B N.OTE
ble1ntinu HL1- HL1- HL1- HL1- HL1- HL1- HL1- HL1- HL1- HL1- HL1- HL1- HL1- HL1- HL1- HL1- HL1-
ao S S S S S S S S S S S S S S S S S
TC FI FI FI FI FI FI FI FI FI FI FI FI FI FI FI FI FI
GBE
TheEvolutionofLINE-1inVertebrates
Table2
LengthandGCCompositionof L1 FamiliesinMammals,Lizard,Frog,and Fish
Total 50UTR ORF1 IGR ORF2 30UTR
Mammals Length 7,144(cid:2)894 1,471(cid:2) 581 1,011(cid:2)82 172(cid:2)222 3,836(cid:2)22 654(cid:2)638
[6,020–9,646] [906–3,229] [891–1,158] [26–719] [3,807–3,882] [148–2,751]
%GC 42.6(cid:2) 1.6 57.2(cid:2)5.8 39.1(cid:2)2.2 36.9(cid:2)4.1 37.9(cid:2)1.3 46.3(cid:2)2.5
[39.2–45.3] [43.3–63.1] [35.9–44.1] [29.7–42.3] [35.5–39.2] [40.7–49.5]
Lizard Clade1 Length 6,435(cid:2)165 1,268(cid:2) 198 1,066(cid:2)21 24(cid:2)48 3,760(cid:2)52 345(cid:2)118
[6,151–6,703] [792–1,465] [762–1,125] [0–110] [3,645–3,813] [194–570]
%GC 36.4(cid:2) 1.0 45.2(cid:2)2.9 37.3(cid:2)2.8 34.5(cid:2)5.7 33.5(cid:2)1.1 33.1(cid:2)5.8
[34.8–37.8] [39.5–49.7] [34.7–43.8] [30.5–38.5] [31.7–35.0] [22.5–40.2] D
o
Clade2 Length 5,381(cid:2)139 229(cid:2)11 981(cid:2) 193 310(cid:2)64 3,737(cid:2)17 125(cid:2)29 wn
[5,234–5,510] [216–238] [762–1,125] [270–384] [3,723–3,756] [96–154] loa
d
%GC 35.1(cid:2) 1.2 44.5(cid:2)1.2 39.7(cid:2)2.1 46.1(cid:2)5.7 32.5(cid:2)0.7 32.6(cid:2)6.7 e
d
[33.6–35.9] [43.1–45.4] [37.3–40.9] [39.6–50.0] [31.7–33.0] [25.3–38.5] fro
Frog Length 5,712(cid:2)234 142(cid:2)18 999(cid:2)83 598(cid:2)263 3,735(cid:2) 159 239(cid:2)81 m
h
[5,470–6,340] [91–162] [873–1,113] [257–1,032] [3,225–3,855] [98–365] ttp
%GC 44.5(cid:2) 3.3 55.2(cid:2)4.4 50.5(cid:2)4.0 43.7(cid:2)4.6 43.3(cid:2)3.2 35.0(cid:2)4.9 s
[38.3–48.1] [44.0–59.5] [42.7–54.2] [33.7–50.1] [37.5–47.0] [25.9–42.3] ://a
c
a
Fish Length 5,794(cid:2)404 165(cid:2)42 877(cid:2)70 536(cid:2)132 3,773(cid:2)71 448(cid:2)471 d
e
[5,380–7,293] [113–267] [780–1,059] [311–730] [3,516–3,837] [167–2,157] m
ic
%GC 36.9(cid:2) 1.3 41.3(cid:2)3.6 47.1(cid:2)2.9 33.7(cid:2)3.2 35.9(cid:2)1.5 24.7(cid:2)4.6 .o
u
[33.9–39.2] [36.0–47.5] [41.0–52.8] [27.7–40.4] [33.3–38.6] [16.7–34.0] p
.c
o
m
/g
b
e
/a
rtic
le
-a
b
s
tra
c
t/8
/1
2
/3
4
8
5
/2
6
8
0
0
4
1
b
y
g
u
e
s
t o
n
1
0
A
p
ril 2
0
1
9
FIG.4.—EvolutionofthemammalianIGR.ThefiguresuggeststhattheancestorofmammalshadanIGRthatwaslostafterthesplitbetweenafrotheria
(elephantandhyrax)andothermammalsandthatanIGRwasregainedinpig.Thebranch-lengthsonthephylogenyarenotuptoscale.
50UTRtendtobeenrichedinGC,relativetootherregionsof contentattheextremitiesoftheelementscontributessignif-
L1.Thisisparticularlytrueinmammals,withanaverageGC icantlytothehigherGCcontentofmammalianL1relativeto
contentof57.2%inthe50UTR.Themammalian30UTRisalso lizardandfish.
GC-rich (46.3%) when compared with other vertebrates, TherearealsoremarkabledifferencesintheGCcontentof
which have remarkably low GC content in this region of L1 theORFs(table2).TheGC-contentofORF1andORF2aresig-
(from24.7%infishto35.0%infrog).Sincemammalshave nificantlydifferentamongvertebrates(ANOVA;F =70.61;
3,51
onaveragelongerUTRsthanothervertebrates,thehighGC- P<0.00001forORF1;F =79.31;P<0.00001forORF2).In
3,51
GenomeBiol.Evol.8(12):3485–3507. doi:10.1093/gbe/evw247 AdvanceAccesspublicationOctober19,2016 3493
GBE
BoissinotandSookdeo
mammalsandlizard,bothORFsshowaconsiderableenrichment fish (12.2) and frog (7.5), suggesting that the transcription
inadenine(42.2%onaverageinORF2and43.4%inORF1), ofL1mightbemoreefficientinfrogandfishthaninamniotes
whichisobservedatthethreecodonpositions(fig.5).Infrog (supplementarymaterialS2,SupplementaryMaterialonline).
andzebrafish,adeninealsotendstobemorefrequentthanthe
otherthreebases(33.7%inORF2and32.1%inORF1),yetthe Evolutionofthe50UTR
differenceisnotaspronouncedasinmammalsandlizard,result-
PreviousworkinmammalshasshownthatL1hastheabilityto
inginanoverallhigherGCcontentoftheORFs(table2).Zebrafish
recruitnovel50UTR,possiblytobypasshostrepressionoftran-
ORF2isuniquebecauseitisenrichedforbothadenineandthy-
scription (Adey, Schichman, et al. 1994; Khan et al. 2006;
mine.Itcanbenotedthatwithineachspeciesthebasecompo-
Sookdeo et al. 2013). We thus decided to examine how
sition, andinparticularthe frequency of adenine, isstrikingly
similar at all codon positions. In all vertebrates, however, the common the replacement of 50UTR across vertebrates is. As Do
w
GCcontentofORF1issignificantlyhigherthanORF2(P<0.05 reportedabove,50UTRsfallintwocategories:thelong50UTR nlo
of mammals and lizard clade 1 and the short 50UTR of fish, a
forallspeciesusingt-test;table2),thelargestdifferencebeing d
e
foundinzebrafish(ORF1=47.1%;ORF2=35.9%). fervoogluatniodnlaizrayrtdrecnladd,es2in.cTehtehseerdeifafreerennocessurbesfltaencttiaanldLi1ff-espreenccifiecs d fro
Considering the differences in base composition in the m
OusRaFgse,.wTaebldee3cidsehdowtos tehxeamcoidnoenhuoswagethfisorreOlaRtFe1s atondcOodRoFn2 i2n00th2e) Bleansgetdhoonf t5h0UeTpRhsylaomgeonnygoefukLa1ry(ofitge.s3()M, wigneocnaeneitnfaelr. https
inalltaxaaswellasanestimatorofthebiasforeachcodon thattheancestralstateismost likelyashort 50UTRandthat ://ac
(RSCU). With very few exceptions, when a codon with an along50UTRevolvedindependentlytwice,intheancestorof ad
e
adenineatthethirdpositionisavailable,itwillbethecodon allmammalsandintheanolelineage. mic
mostfrequentlyused.ThisistrueforbothORFsandinalltaxa. Using DOTMATCHER we compared the 50UTRs of mam- .o
u
This was further confirmed by calculating two estimators of mals but we failed to find any significant similarity among p.c
codonusagebias,CAIandNc.CAIcomparesthecodonusage them, which is consistent withthe rapid50UTRturnover de- om
of a gene of interest (ORF1 and ORF2 in our case) with the scribed in primates and rodents (Adey, Schichman, et al. /gb
e
codonusageofthehost’sgenomewhereasNcestimateshow 1994; Khan et al. 2006; Sookdeo et al. 2013). Despite the /a
the codon usage differs from equal usage of synonymous absenceofhomologyamong50UTRs,almostallmammalian rticle
codons (table 4). The elevated values of Nc are consistent L1sbeginwithasequenceofconsensusG2-6(A/C)G2AGNCA -ab
with a substantial codon usage bias, yet none of the values AGATGGCGGA,themotifCAAGATGGCcorrespondingtoa stra
ofCAIweresignificantlydifferentfromexpectationgiventhe YY1transcriptionfactorbindingsitewhichiscriticalfortran- ct/8
codon usage of the host and the GC content of the genes. scription initiation (Athanikar et al. 2004). The only excep- /1
2
Thisisnotreallysurprisingsincetheenrichmentinadenineis tionsarethemouseandratelementswhichdonotstartwith /3
4
foundatallthreecodonpositions. the YY1 binding site but it was shown that the monomers 85
Weexaminedhowdifferencesinbasecompositionaffect constitutive of their 50UTR contain signals for transcription /26
8
theaminoacidcompositionoftheORFs(fig.6).Twoobser- initiation (Adey, Tollefsbol, et al. 1994). Mammalian 50UTRs 00
4
vationscanbemade.First,mammalianandlizardL1areen- have very high GC content (from 51.9% to 61.6%), with 1
b
richedinlysineandglutamicacid,twoaminoacidsencoded twonotableexceptions:thecow(43.3%)andthearmadillo y g
byA-richcodons(AAAandAAGforlysineandGAAandGAG (46.4%), which also happen to have the longest 50UTRs in ue
s
for glutamic acid), and for both amino acids it is the A-rich mammals(3,229and2,029bp,respectively).Allmammalian t o
codon that is strongly preferred (AAA and GAA; table 3). 50UTRsareenrichedinCpGdinucleotides,whichareforming n 1
0
Second,thefrogandfishL1scontainahigherproportionof CpG islands. The average number of CpG dinucleotides is A
p
tchorneteainamaninoA:aacliadnsineenc(GodCeAd,GbyCCco,dGoCnGs,thGaCtUa)r,eplreoslsinleik(eClyCAto, 6re2g.i1onanodf tvhaerie5s0UfTroRmth3at1fiitnsothpeosdseufimnittioon9o4fina ChpoGrsei.slaTnhde ril 20
1
CCC,CCG,CCU),andSerine(AGC,AGU,UCA,UCC,UCG, alwaysresideatthe50 extremityoftheUTR,withtheexcep- 9
UCU). These differences in amino acid composition are ob- tionofthecow(whoseCpGislandbegins~250bpfromthe
servedforbothORFsandarethusunlikelytoresultfromse- 50 extremity). A number of 50UTRs contain motifs (~70–100
lectiononthefunctionoftheproteins. bp) that are tandemly duplicated two (rat, hyrax), three
OneoftheconsequencesofanenrichmentinA-richcodon (horse, elephant, opossum), or many (mouse) times (fig. 7).
isthepotentialformationofprematurepolyadenylationsignal OtherelementsaredramaticallyenrichedinG-rich(pig)orT-
and thus inefficient transcription (Perepelitsa-Belancio and rich (dog, cow, armadillo) low-complexity repeats, the cow
Deininger2003).Weestimatedtheaveragenumberofcanon- presenting the most extreme examples since it contains a
ical and non-canonical (AATAAA, ATTAAA) poly-adenylation ~840-bpregioncomposedexclusivelyofT-richshortrepeats
signals in the ORFs. As expected, the number of potential (fig. 7).
premature poly-adenylation signals in the ORFs is larger in Thoughsimilarinlength,thelong50UTRsofthelizardclade
mammals (16.3 on average) and lizard (25.0) than it is in 1differfromthemammalian50UTRsinseveralrespects.The
3494 GenomeBiol.Evol.8(12):3485–3507. doi:10.1093/gbe/evw247 AdvanceAccesspublicationOctober19,2016