Origin of genes with unresolved ancestry

56 Pages·2013·3.82 MB·English

Master in Proteomics and Bioinformatics
Origin of genes with
unresolved ancestry
Analysis of orphan genes in
H.sapiens, D.melanogaster and S.cerevisiae
Adrián César Razquin
Thesis supervisors:
Dr. Evgenia Kriventseva
and
Prof. Evgeny Zdobnov
Université de Genève
January 2013

CONTENTS
Abstract .................................................................... 3
1 Introduction .............................................................. 5
1.1 Ontheoriginofgenes ................................................... 5
1.2 Orthology ............................................................. 9
1.2.1 Homologs,orthologsandparalogs ..................................... 9
1.2.2 Orthologyinferencemethods ......................................... 10
1.2.3 Orthologydatabases. OrthoDB. ....................................... 11
1.3 Orphans .............................................................. 12
1.4 Aimoftheproject ...................................................... 14
2 Methods ................................................................ 15
2.1 Programminglanguage ................................................. 15
2.2 Identificationoforphans ................................................ 15
2.3 Correlationofnumberoforphansandevolutionarydistance ................ 15
2.4 GO-terms ............................................................. 16
2.5 Evolutionaryrates ..................................................... 17
2.6 Siblinggroups ......................................................... 17
2.7 TransposableElements ................................................. 18
3 Results .................................................................. 19
3.1 Identificationandcharacterizationoforphans ............................. 19
3.1.1 Identification ....................................................... 19
3.1.2 Numberoforphansandevolutionarydistances ......................... 19
3.1.3 GeneOntologyanalysis. ............................................. 21
3.1.4 Orphansasfast-evolvinggenes ....................................... 22
3.2 Analysisofsiblinggroups ............................................... 22
3.3 Analysisoftransposableelements ........................................ 25
3.4 Casestudy ............................................................ 28
4 Discussion .............................................................. 33
Appendix .................................................................. 37
Acknowledgements ......................................................... 47
References ................................................................. 49

Abstract
Since the very beginning of comparative genomic studies, researchers became aware of
the existence of a set of genes that shows no clear homology when compared with other
genomes. Althoughtheywereinitiallyattributedtothelackofavailablegenomicdata,these
orphangeneshavebeenidentifiedinallgenomeprojectstodateandtheymightconstitutean
irreduciblefractionofgenespresentinthegenomeofeveryorganism. Differenthypotheses
have been proposed in the last years to explain the evolutionary origin of orphans, but the
issueremainsstillunclear.
The present study identifies and characterizes orphan proteins in H.sapiens, D.melano-
gasterandS.cerevisiaebasedontheorthologyclassificationofOrthoDBandaimstoprovide
an insight into some possible origins of orphans. We show that orphans constitute a large
fractionoftheproteomesofthethreeselectedspecies,andweprovideevidenceoftheirhigh
evolutionaryrateandtheirrelationtoadaptivefunctions. Moreover, basedontheanalysis
of homology with non-ortholog proteins, we suggest that most of them might have orig-
inated by duplication followed by an accelerated evolution, alone or in combination with
otherprocessesinvolvingdomainrearrangementssuchasexonshuffling. Finally,weshow
that a small percentage of the orphans contains sequences derived from transposable ele-
ments (TEs) in their coding regions, which might have also contributed to their origin and
divergence.
3
4
1
Introduction
1.1 On the origin of genes
Sincethepublicationofthefirstwhole-genomesequenceofafree-livingorganismin1995
[1],afastincreasingnumberofgenomesissequencedandpublishedeveryyear. Asanillus-
tration,theGenomesOnLineDatabase(GOLD),aworldwideresourceformonitoringgenome
andmetagenomeprojects, containedinformationfor11472sequencing projectsinSeptem-
ber 2011, of which 2907 had been completed –either finished or as permanent drafts-. The
statistics since 1995 show an approximately exponential growth in the number of genome
sequencingprojects[2].
Theanalysisofgenomicdatahasrevealedagreatvariationingenenumberamongorgan-
isms,indicatingtheexistenceofacontinuousprocessofgeneorigination. Severalmolecular
mechanismscanbeinvolvedintheformationofnewgenes,eithercreatingthemfrompre-
existingonesorpartsofthem[3]orde-novofromnoncodingsequences. Inanycase,thenew
gene must appear in the germ line in order to be transmitted to the offspring and spread
withinthepopulation,whereitcanfinallyhaveoneoftwomajoroutcomes: fixationorex-
tinction[4].
HereIbrieflyreviewthemaincharacteristicsofprocessescreatingnovelgenes.
I. Geneduplication. Geneduplicationwasthefirstmechanismproposedforgeneorigina-
tion, initially suggested by Muller and Haldane in the 1930’s [5 and 6]. However, it was
mainlythankstoOhno’sworkinthe1970s[7]thatgeneduplicationstartedtoberegarded
asthemostimportantforcegeneratinggenenovelty.
Two major types of duplications can take place in the genome of an organism: seg-
mentalduplications,whichincludeindividualgenesorgenomicregions,andwhole-genome
duplications (WGD), which consist in the copy of the entire genome. WGD are rare but
recurrenteventsthatcauseenormousgenomicchange,abletoleadtothere-diploidization
ofagenome–e.g. plantssuchasTriticumaestivum,thebreadwheat,whichishexaploid-.
Thisprocesshasbeendescribedinbothprokaryotes[8]andeukaryotes,withwell-known
caseshavingoccurredinyeast,angiospermsandteleosts[9]. UptotworoundsofWGD
arethoughttohaveoccurredbeforetheoriginofthevertebratelineage[10]. Mechanisms
underlyingWGDeventsincludegenomedoublingandgameticnon-reduction,whichrepre-
sentcellcyclefailuresduringmitosisandmeiosis,respectively,andpolyspermia[11].
Segmentalduplications,ontheotherhand,occurmorefrequentlythanWGDbutaffect
smallerregions,rangingfromafewnucleotidestoseveralthousandkilobases. Twomole-
cularmechanismsthatcanleadtosegmentalduplicationsarenon-allelichomologousrecom-
bination(NAHR)andillegitimaterecombination(IR)[12],whichcanberegardedaserrors
innormalcellularprocesses. Inthissense, NAHR-alsocalledunequalcrossing-over-isa
recombination event that takes place between highly similar but non-allelic sequences,
for example due to the missalignment of repeated elements like low copy number repeats
(LCRs)ortransposibleelements(TEs);andIRresultsfromthemalfuctionofthenon-homol-
ogousend-joining(NHEJ)pathwayfordouble-strandDNAbreak(DSB)repair, whichfuses
twofreeDNA-ends[13]. SomestudiesinhumananddrosophilaconcludedthatNAHR
5
Introduction
is a frequent mechanism generating segmental duplications, which mainly creates dis-
persed duplicates, while IR is more rare and plays a more important role in producing
tandemduplications[14and15]. Homeobox[16]andglobin[17]genesarewell-knownex-
amplesofsegmentalduplications. Anothersourceofduplicationsisretroposition,which
createsduplicategenesinnewgenomiclocationsbyreversetranscriptionofmaturemR-
NAs, recruiting the necessary enzymatic activities from autonomous transposable ele-
ments such as the L1 elements in mammals. The copies resulting from retroposition
differ from regular gene duplicates in several features, such as their lack of introns and
thepresenceofflankingshortdirectrepeats. Besides,aspromoterregionsareusuallynot
retroposedfromtheoriginalgene,thesegeneduplicatesbecomenon-expressedprocessed
pseudogenesunlesstheyrecruitnewregulatorysequencesfromneighboringgenes-which
dependsonthegenomiclocationofthenewcopy-. [3]
Initsmodel,Ohnoconsideredthatmostofthenewgenesoriginatedfromaduplica-
tioneventwouldrapidlyaccumulatemutationsanddegenerateintheformofapseudo-
gene[18]. Nevertheless,nowadaysitisknownthatevenifalotofgeneduplicatesbecome
pseudogenes, many of them can be fixed and remain functional [19] through processes
such as evolution of redundancies, subfunctionalization and neofunctionalization [12]. Evolu-
tionofredundancieshappenswhenthegenedosageincreasecausedbyaduplicationisfa-
voredbynaturalselection,andthereforethetwogenecopies,whichperformequalfunc-
tions,coexistunchangedinthegenome. Thisfatehasbeenespeciallydescribedinyeast
[20]. Subfunctionalization, also called the duplication-degeneration-complementation (DDC)
model[19,21and22],istheprocessofpartitioningtheancestralgenefunctionbetween
the two copies, mainly by complementary degeneration of regulatory elements. In this
case, the union of activities and expression patterns of the two duplicates results in the
original function of the ancestral gene. A well known example of this process are the
gene duplicates GAL1/GAL3 of Saccharomyces cerevisiae [23]. Finally, neofunctionalization
consists in the acquisition of novel functions by one of the gene copies while the other
copy retains the ancestral function. Indeed, as the original gene function is ensured by
oneofthecopies,theotherduplicatecanbesubjecttoamorerelaxedpurifyingselection
andthereforeitcanrapidlyaccumulatemutationsthatinotherconditionswouldbeelim-
inated. Thisacceleratedevolutionnotonlycanincludenucleotidesubstitutions,butalso
largermodificationssuchasinsertionsanddeletions,domainshuffling,fussionsandfis-
sions. Eventually,mutationswouldleadtoanewallelebearingabeneficialfunctionfor
theorganism, whichwouldbepreservedbyselection. Severalworkshavereportedthe
influenceofadaptivenaturalselectionintheevolutionofyounggeneduplicates[24and
25]. As an illustration, many proteins from the Major Histocompatibility Complex (MHC)
are known to have originated from gene duplication and subsequent neofunctionaliza-
tion[26].
Furthermore,thenatureoftheduplication-segmentalorWGD-,canhaveadirectin-
fluenceonthefateofthenewborngenes. Asanillustration,asegmentalduplicationof
agenewhoseproductpresentsmanyinteractionpartners-e.g. componentsofsignaling
pathways,ortranscriptionalregulators-woulddisruptthestoichiometryofthecomplex,
andmostprobablytheduplicationwouldbecounter-selected. Contrarily,iftheduplica-
tionresultsfromaWGDevent,thegenedosagebalancewouldbemaintained,asallthe
interactors of the network would be duplicated, and therefore the duplication could be
retained[27].
Around 15% of the human genes are thought to have arisen by duplication, as well
as8-20%ofthegenesofDrosophilamelanogaster,Caenorhabditiselegans,andSaccharomyces
cerevisiae [11]. Moreover, frequences of gene duplications were estimated at more than
6
Introduction
100 gene duplications per 1 million years in the human genome [28] and 17 genes per
millionyearsinflies[29].
II. Exon shuffling and gene fusion and fission. Exon shuffling, and to a smaller extent gene
fusionandfission,areprocessesthatgeneratenewexonarrangementsthatcanleadtothe
creationofnovelchimericgenes.
Exon shuffling, or intron-mediated recombination, is the process through which a new
exonisinsertedintoanexistinggeneoranexonisduplicatedwithinagenetogenerate
anewintron-exonstructure[3]. Thismechanismforgeneratingnewgeneswasinitially
proposedin1978byWalterGilbert[30],andnowadaysisthoughttobeoneofthemajor
evolutionaryforcesthatshapeeukaryotegenomesandproteomes. Around19%ofexons
ineukaryoticgenesareestimatedtohavebeencreatedbyexonshuffling[3]
Thesamemechanismsthatproducesegmentalduplicationscanleadtotheectopicre-
combinationofexons: non-homologousorillegitimaterecombination,andretroposition. Con-
sequently, exon shuffling seems to have a huge importance in the evolution of complex
genomes,asthesecontainmanyintronsandrepetitiveelementsthatfacilitateintronicre-
combinationevents[31]. Also,exonshufflingappearstohavebeenespeciallyimportant
intheformationofmultidomainproteinsinmetazoans[32and33].
Two features of genomes are used to evaluate exon shuffling: intron phase and exon
symmetry. Intronphaseisthepositioninwhichtheintroninterruptsthereadingframeof
agene: phase0intronsareplacedbetweencodons,whereasphase1andphase2introns
lie after the first and the second nucleotides of the codon, respectively. Exon symmetry
classifies exons in nine categories according to the phase of the introns sourrounding
them. Exonsaresymmetricifbothintronphasesareequalandassymmetricotherwise.
The importance of this classification resides in the fact that only symmetric exons can
be inserted, duplicated or deleted in introns -of the same phase- without changing the
readingframe, andthereforetheseeventswouldbelessaffectedbypurifyingselection.
Accordingly,researchershavedetectedanexcessofsymmetricexonsinmosteukaryotic
genomesanalyzedtodate,mainlyoftype1-1inmetazoanspeciesand0-0exonsinnon
metazoans. Thisfactsuggeststheimportanceofexonshufflinginmodelinggenomes[33
and34].
Gene fusions and fissions have been mainly described in the origin of many genes in
prokaryotic genomes [35], although examples in eukaryotes also exist –e.g. the fusion
geneKUA-UEVinhumans[36]-. Kummerfeldetal. estimatethatfusionsarearoundfour
timesmorefrequentthanfissions,probablybecausetheyimplythedeactivationorloss
of certain gene regions, which is genetically simpler to achieve than the gain of these
same regions necessary for a fission event. These are mainly terminal sequences of the
firstgenesuchastranslationstopcodonsortranscriptionterminationsignals,andinitial
regulatoryregionsofthesecondgene,likepromoterregions[37].
III.Transposable elements and lateral gene transfer. Transposable elements (TE) are another
veryimportantsourceofgenenovelty. Indeed,sincethediscoveryof“jumpinggenes”by
theNobelLaureateBarbaraMcClintockinthe1950’s[38and39],theknowledgeandun-
derstandingofmobileelementshasdramaticallyincreasedandevolvedinthescientific
community. Frombeingregardedjustas“junk”or“selfish”DNA,thecurrentviewconsid-
ersTEsasoneofthemajorcomponentsofgenomes,whichplayaveryimportantrolein
evolutionbypromotingandcontrollingprocessessuchaschromosomalrearrangements,
geneexpression,orevenpopulationadapationandspeciation[40].
7
Introduction
TEs are pieces of DNA with the capability of changing their genomic location, very
often making a duplicate copy of themselves during the process. The current most ac-
ceptedclassificationofTEs[41–43]dividesthemintwomainclasses,dependingonthe
natureofthetranspositionintermediate: Class-Itransposons,or“retrotransposons”,usean
RNAintermediateandreversetranscriptionina“copy-and-paste”manner,andClass-II
transposons,or“DNAtransposons”,useanDNAintermediate,mainlyby“cut-and-paste”.
Theseclassesarethenfurthersubdividedintosubclasses,orders,superfamiliesandfam-
iliesbasedonmechanistic,enzymaticandstructureandsequencesimilaritycriteria. The
traditional view separated Class-I tranposons into LTR retrotransposons, which contain
flanking direct Long Terminal Repeats (LTRs) and derive from retroviruses, and non-LTR
retrotransposons,whichlacktheseLTRs. Twowell-knowngroupsofnon-LTRreptrotrans-
posons are LINEs -for Long INterspersed ELements- and SINEs -Short INterspersed ELe-
ments-, different mainly by their lengths and their coding capacity. Moreover, some of
thesegroupsofTEsareautonomousbydefinition,meaningthattheyencodetheneces-
saryproteinsforthetranpositionprocess-itisthecaseofDNAtransposons, LTRs, and
LINEs- and others, such as SINEs, are non-autonomous, because they lack ORFs and
therefore depend in trans on other TEs for mobilization. Nevertheless, a large extent of
anyorganism’sTEsaredefectiveandhavelosttheirmobilizationcapacity,henceremain-
inginactive.
The systematic analysis of genome sequences has shown a great species to species
variability in TE content. As an illustriation, TEs make up 80% or more of most plant
genomicDNA,butvarybetween3and20%infungiandbetween3and45%inmetazoa.
Around 44% of the human genome is made of TEs, and although all the main classes
are present, non-LTR retrotransposons -LINEs and SINEs- are predominant and make
togethermorethan70%ofhumanTEs. Drosophila’sTEcontentfallsaround18%,andno
SINEs are found. Also, this genome seems to contain many young and active families.
Ontheotherhand,theyeastS.cerevisiaecontainsonlyLTRtransposons-theso-calledTy
elements-,whichconstituteintotalnomorethan5%ofitsgenome[40and41].
ThecapacityofTEstoevolvegenomicfunctionsisnowdaysawell-documentedphe-
nomenon,whichisusallyreferredasTE“domestication”or“neofunctionalization”[44and
45]. ByexonizationfromTEsequences,newgenesorpartsofgenescanbecreated[45].
Asanillustration,Aluelements,atypeofSINEsthatmakeupmorethan10%ofthehu-
mangenome,arefrequentlyexaptatedtocreatenewexonsinprimategenomes,aprocess
thathasbeenestimatedtoconstitutemorethan50%ofnewexonsinH.sapiens. Thisca-
pacityhasbeenattributedtotheexistenceofmultiplesitesintheAluelementsthatare
similartosplicesites-pseudosplicesites-andcanbecomeactivebyaccumulationofmuta-
tions[46]. Accordingly,4%ofhumangenesareestimatedtocontainTEmotifsinintheir
codingsequences[31]. Somewell-knownexamplesofgenescreatedbyexonizationfrom
TEsaretheRAGgenesoftheimmunoglobulinsystem,originatedfromDNAtransposons
[47]ortheTARTandHeTelementsinDrosophila,whichactastelomerases[48].
LateralorHorizontalGeneTransfer(HGT)referstothetransmissionofgeneticmaterial
between different organisms, in contraposition to the usual parent-to-offspring vertical
inheritance. HGT is common in bacteria and it has long been recognized to be a major
forcedrivingprokaryoteevolution[49]. Nevertheless,ithasbeenshownthatTEscanbe
horizontally transferred between eukaryotic species, therefore indicating that HGT can
also play an important role in generating genomic variation and innovation in eukary-
otes. Probably the most well-known case of a HGT event between eukaryotes are the
transferofthePelementsofDrosophila,afamilyofTEsthatescapedfromD.willistoniand
colonized the genome of laboratory strains of D.melanogaster [50]. Schaack et al. identi-
fied 218 convincing cases of HGT events in different eukaryotic lineages -plants, fungi,
8