Hyper-eccentric structural genes in the mitochondrial genome of the algal parasite Hemistasia ...

29 Pages·2016·2.46 MB·English

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Hyper-Eccentric Structural Genes in the Mitochondrial
Genome of the Algal Parasite Hemistasia phaeocysticola
Akinori Yabuki1,*, Goro Tanifuji2,3, Chiho Kusaka1, Kiyotaka Takishita1, and Katsunori Fujikura1
1JapanAgencyforMarine-EarthScienceandTechnology(JAMSTEC),Yokosuka,Kanagawa,Japan
2GraduateSchoolofLifeandEnvironmentalSciences,UniversityofTsukuba,Tsukuba,Ibaraki,Japan
3Presentaddress:DepartmentofZoology,NationalMuseumofNatureandScience,Tsukuba,Ibaraki,Japan
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*Correspondingauthor:E-mail:[email protected]. w
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Accepted:August 20, 2016 loa
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Datadeposition:ThisprojecthasbeendepositedatGenBank/EMBL/DDBJundertheaccessionDRA003989(PacBiosequencingdataforthe d
mtDNAofHemistasiaphaeocysticola)andLC114081-LC114090&LC120367-LC120387(themitochondrialgenomicDNAandcDNAsequences fro
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obtainedbytheSangermethod). h
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Abstract a
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Diplonemidmitochondriaareconsideredtohaveveryeccentricstructuralgenes.Codingregionsofindividualdiplonemidmitochon- ic
.o
drialgenesarefragmentedintosmallpiecesandfoundondifferentcircularDNAs.ShortRNAstranscribedfromeachDNAmolecule u
p
maturethroughauniqueRNAmaturationprocessinvolvingassemblyandthreetypesofRNAediting(i.e.,UinsertionandA-to-Iand .co
m
C-to-Usubstitutions), although themolecular mechanism(s) of RNA maturation and the evolutionary history of these eccentric /g
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structuralgenesstillremaintobeunderstood.Sincethegenefragmentationpatternisgenerallyconservedamongthediplonemid e
/a
speciesstudiedtodate,itwasconsideredthattheirstructuralcomplexityhasplateauedandfurthergenefragmentationcouldnot rtic
occur.Here,weshowthemitochondrialgenestructureofHemistasiaphaeocysticola,whichwasrecentlyidentifiedasamemberofa le
-a
novellineageindiplonemids,bycomparisonofthemitochondrialDNAsequenceswithcDNAsequencessynthesizedfrommature b
s
mRNA. The genes of H. phaeocysticola are fragmented much more finely than those of other diplonemids studied to date. tra
c
Furthermore,inadditiontoallknowntypesofRNAediting,itissuggestedthatanovelprocessingstep(i.e.,secondaryRNAinsertion) t/8
isinvolvedintheRNAmaturationinthemitochondriaofH.phaeocysticola.Ourfindingsdemonstratethetremendousplasticityof /9/2
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mitochondrialgenestructures. 7
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Key words: gene fragmentation, RNA processing, mitochondrial genome, diplonemids, Euglenozoa. 6
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Introduction y
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Gene coding regions are often interrupted. Introns are the Diplonemidsareunicellularflagellatesthatarefree-livingor s
primary structural interrupters of coding region, such as parasitize algae and invertebrates (Lukesˇ et al. 2015). While t on
spliceosomal introns, group I and II introns, and archaeal thediversityofdiplonemidsremainsincompletelyunderstood 28
M
introns (Moreira et al. 2012). On the other hand, some (Lara et al. 2009; de Vargas et al. 2015), the fragmented a
genes of microbial eukaryotes, such as ciliates and apicom- genesfoundinthediplonemidmitochondrialgenomeshave rch
2
plexans, are further fragmented and found at multiple loci beenstudiedintensively;theircomplexandunusualstructures 0
1
in genomes. These fragmented genes may result in discon- haveattractedattentioninthefieldofnotonlyorganellege- 9
tinuous products that work as subunits or split proteins nomics,butalsoRNAprocessing.Themitochondrialgenomes
(Heinonen et al. 1987; Edqvist et al. 2000; Feagin et al. of diplonemidsstudiedto date, namely Diplonema spp. and
2012). Among the known gene structures, the most com- Rhynchopus euleeides, comprise multiple mini-circular chro-
plex and unusual are considered to be those of the mito- mosomes~5–12kb(Burgeretal.2012).Althoughitstillre-
chondrial genome of diplonemids (Euglenozoa), a sister mains unknown how many genes there are in the
group of kinetoplastid flagellates (Marande et al. 2005; mitochondrial genomes of diplonemids, the coding regions
Vlcek et al. 2011). of all genes studied to date including the rRNA genes are
(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]
2870 GenomeBiol.Evol.8(9):2870–2878. doi:10.1093/gbe/evw207 AdvanceAccesspublicationAugust25,2016
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Hyper-EccentricStructuralGenesintheMitochondrialGenomeoftheAlgalParasiteHemistasiaphaeocysticola
fragmentedintosmallpiecescalled“modules”;nononfrag- steps for the removal of the 50 phosphates and the 50 cap
mentedversionofthegenehasbeenfound(Vlceketal.2011; structure were skipped in cDNA synthesis using the
Valachetal.2014).Forexample,theprotein-codingregionof GeneRacerKit.
cox1 is fragmented into nine pieces, and its fragmentation
patternisgenerallyconservedamongthediplonemidspecies Sequencingofcox1cDNAandChromosomesPossessing
studied to date (Marande and Burger 2007; Kiethega et al. cox1Modules
2011); the number of modules and fragmented positions
Polymerasechainreaction(PCR)wasperformedusingHotStar
foundincox1aregenerallythesameamongfourdiplonemid
Taq(Qiagen),andPCRproductswereclonedintothepCR2.1
species sequenced to date. Each module is independently
vector usingaTOPO TACloning Kit(LifeTechnologies). The
transcribed, and primary transcripts undergo secondary as- cloneswerecompletelysequencedonbothstrands.Thepar- D
o
sembly into a single RNA (Marande et al. 2005; Marande w
tial sequence of cox1 cDNA was amplified from the synthe- n
andBurger2007;Kiethegaetal.2013). lo
sized cDNA using the dip cox1 F and dip cox1 R primer set a
ThreetypesofRNAediting[i.e.,uridine(U)insertionandA- (supplementary table S1, Supplementary Material online). ded
to-IandC-to-Usubstitutions] arealsoinvolvedinthemRNA Amplification consisted of 35 cycles of denaturing at 94(cid:2)C fro
maturation process before the end products of assembled for30s,annealingat52(cid:2)Cfor30s,andextensionat72(cid:2)C m h
RNA sequences are translated to the proper amino acid se- for 1 min. Based on this partial sequence, we designed six ttp
s
quences (Marande and Burger 2007; Kiethega et al. 2013; primers (p1–p6) (supplementary table S1, Supplementary ://a
Moreira et al. 2016). As misassembled mRNA, in which the c
Material online), and the complete cDNA sequence of a
d
short RNAs are assembled in incorrect order, has not been e
mature cox1 mRNA was amplified using these primers and m
documented, this RNA maturation process is considered to adapterprimersfromthecDNAsynthesizedbytheGeneRacer ic.o
progress quite accurately (Burger et al. 2012). However, the u
kit.ThePCRprogramforfull-lengthcox1cDNAconsistedof p
underlying molecular mechanism(s) remain(s) unknown. 35cyclesofdenaturingat94(cid:2)Cfor30s,annealingat55(cid:2)C .co
m
Althoughitisunclearhowandwhytheseunusualgenestruc- for30s,andextensionat72(cid:2)Cfor2min.ThePCRconducted /g
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tures were established, they have been suggested to be a with genomic DNA and three combinations of outward e/a
resultofconstructiveneutralevolution;ithasalsobeensug- primer sets (i.e., p1 and p2, p3 and p4, and p5 and p6) for rtic
gestedthattherearenofunctionaladvantagesofgenefrag- theamplificationofchromosomalsequencesconsistedoftwo le-a
mentation (Flegontov et al. 2011). Furthermore, because of continuoussteps:(1)threecyclesofdenaturingat94(cid:2)Cfor30 bs
thesharedmannerofgenefragmentationamongdiplonemid s,annealingat58(cid:2)Cfor30s,andextensionat72(cid:2)Cfor2.5 trac
speciesexaminedtodate,thecomplexityoftheirgenestruc- min;(2)32cyclesofdenaturingat94(cid:2)Cfor30s,annealingat t/8
/9
ture may have plateaued during evolution and further gene 55(cid:2)Cfor30s,andextensionat72(cid:2)Cfor2.5min.ThePCRto /2
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fragmentation may have not occurred in this lineage confirm the sequence variation and existence of immature 70
(Flegontovetal.2011). mRNA was conducted with the primer set, “cox1 FULL F” /26
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Hemistasiaphaeocysticolawasrecentlyidentifiedasarep- and“cox1FULLR”;theprogramincluded25cyclesofdena- 98
resentativeofalargeenvironmentalcladewithindiplonemids, turing at 94(cid:2)C for 30 s, annealing at 55(cid:2)C for 30 s, and 50
b
and its phylogenetic position is distinct from that of other extensionat72(cid:2)Cfor2min.TheacquiredcDNAsequences y g
diplonemidswhosemitochondrialgenestructureshavebeen were manually aligned and the structure of the cDNA se- ue
s
analyzed (Yabuki and Tame 2015). In the present study, we quencesthatwerederivedfrompossibleimmatureandmis- t o
show the hyper-eccentric degree of the mitochondrial gene assembled cox1 mRNA (i.e., LC114084–LC114087) was n 2
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structureofH.phaeocysticolawithcomparisontomitochon- predicted by comparison with the cDNA sequence derived M
drialgenesinotherdiplonemids.Wealsodiscussthepossibil- frommaturemRNA(i.e.,LC114081). arc
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ityofauniqueRNAmaturationprocessandtheevolutionary 2
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constraintonthegenestructureinH.phaeocysticolamtDNA. SouthernHybridizationAnalysis 19
Total genomic DNA from H. phaeocysticola was prepared
Materials and Methods using a standard cetyltrimethylammonium bromide (CTAB)
protocol (Doyle and Doyle 1990). Southern hybridization
DNAandRNAExtractionandcDNAPreparation
probes targeting the shared region, the distinct noncoding
Total genomic DNA and RNA were extracted from ~100 ml region of the chromosomes possessing modules 13-1 and
samplesof2-week-oldcultures(NIES-3356)usingtheDNeasy 13-2, were amplified using the following primers: “CONS
Plantminikit(Qiagen)andTRIzol(LifeTechnologies),respec- probe F,” “CONS probe R,” “M13-1 probe F,” “M13-1
tively. cDNA was synthesized from DNase-treated RNA by probeR,”“M13-2probeF,”and“M13-2probeR”(supple-
SuperScript II (Life Technologies) using random hexamers or mentary table S1, Supplementary Material online). The PCR
by the GeneRacer Kit (Life Technologies). As the RNA tran- productswereclonedintothepGEMT-easyvector(Promega)
scribedinmitochondriadoesnothavea50 capstructure,the and used as templates for digoxygenin (DIG)-labeled probe
GenomeBiol.Evol.8(9):2870–2878. doi:10.1093/gbe/evw207 AdvanceAccesspublicationAugust25,2016 2871
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Yabukietal.
synthesis according to the manufacturer’s instructions (PCR sequencesweresearchedforusingthecompletecDNAse-
DIG labeling kit, Roche Diagnostics). The 200 ng of total quencesofeachgeneasaqueryusingblastn.Thebound-
DNAperlanewereappliedforelectrophoresisandtransferred ary positions between neighboring modules were
toapositivelychargednylonmembranebycapillarytransfer. estimated by eye. The coding regions that could not be
SouthernhybridizationwascarriedoutinDIGEasyHybbuffer detectedevenbyblastnwiththeoptionforshortsequence
(RocheDiagnostics)withtwoml/mlDIG-labeledprobesover- search (i.e., “-task blastn-short”) were left as missing re-
nightat37(cid:2)C.Themembraneswererinsedtwicefor10min gionsinthisstudy,buttheundetectedregionscomposedof
at room temperature in low-stringency buffer (0.1% SDS, onlyTresidueswereregardedasthesitesforuridine-inser-
2(cid:3)SSC),followedbytwo15-minrinsesat65(cid:2)Cinhigh-strin- tion RNA editing. The G/T content of each missing region
gency buffer (0.1% SDS, 0.5(cid:3)SSC). Hybridization signals was calculated manually. It was here assumed that each D
were detected according to standard procedures in the DIG missing region exists as a single module in the mitochon- ow
n
detectionkit(RocheDiagnostics). drialgenomeandtentativemodulenumberswereassigned lo
a
tothem,respectively. de
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MitochondrialGenomeSequencing fro
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The mitochondrial chromosomes were independently ampli- Results h
fied from the total DNA using two primer sets—“CONS. fR ttp
s
andCONS.rF1”and“CONS.fRandCONSrF2”—toreduce PreliminarySurveyofcox1-CodingChromosomesand ://a
theriskofbiasedamplification.Notethattheseprimerswere DetectionoftheGeneral“Core” ofMitochondrial ca
d
designedtooutwardlyannealwithinthesharedregionthatis Chromosomes e
m
possiblyconservedamongallchromosomes(seebelow).Each ic
ThepartialcDNAofthematurecox1mRNAofH.phaeocys- .o
amplificationconsistedof40cyclesofdenaturingat94(cid:2)Cfor ticola was obtained by reverse-transcription polymerase up
30s,annealing,andextensionat72(cid:2)Cfor3.5min.ThePCR chain reaction (RT-PCR) using “dip cox1 F” and “dip cox1 .com
products contained sequences of various length and more R”(supplementarytableS1,SupplementaryMaterialonline). /gb
than 100 clones were randomly sequenced to check that e
No product with the same primers was amplified from ge- /a
the amplification was successful (it should be noted that nomicDNA. ThecompletecDNAsequenceofmaturecox1 rtic
thesecloneswerenotsequencedcompletely).ThePCRprod- le
mRNAwasthendeterminedby50and30RACE(depositedin -a
uctsweredividedintotwogroups:1-2and2-20kb,andthen b
directlyutilizedasinitialmaterial(withoutanyfragmentation GenbankasLC114081)(fig.1A).Theaminoacidsequence stra
was translated from this cDNA sequence under the rule of c
process) for the sequence library of PacBio RSII. Hence, two t/8
genetic code table 4 (i.e., The Mold, Protozoan, and /9
librarieswereconstructedusingtheSMRTbellTemplatekit1.0 Coelenterate Mitochondrial Code and the Mycoplasma/ /28
(Pacific Biosciences). The mitochondrial genome library con- 7
Spiroplasma Code), but not under the standard code table 0
struction and sequencing by PacBio RSII (Pacific Biosciences) /2
(http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi, 66
were performed at the Dragon Genomics Center (TAKARA 9
lastaccessed7September2016).Itwasalsoconfirmedthat 8
Bio).Thelibrariesconstructedfromthe1-2-and2-20-kbPCR 50
theaminoacidsequencesofcox1inthisstudyclusterwith b
productsweresequencedbyoneandtwoSMRTcells,respec- y
otherdiplonemidcox1sequenceswith100%supportinthe g
tively. The circular consensus sequencing (CCS) reads were u
phylogenetic tree (supplementary material, Supplementary es
generatedfromrawreadsbytheDragonGenomicsCenter. Materialonline). t o
n
Five mitochondrial chromosomes that possess modules 28
CompletecDNASequencingofcob,cox2,andnad7 M
numbered 7, 12 (two types), and 13 (two types) were ac-
a
Theprimersfor30and50RACEweredesignedonthebasisof quiredbythegenomicPCRconductedusingoutwardprimers rch
themodulesequencesofcox2,cob,andnad7detectedinthe annealingtovariousfragmentsofthededucedcodingregion 20
1
preliminary mitochondrial genome sequencing (supplemen- (fig. 1A and B). The mitochondrial chromosome sequences 9
tary tableS1, SupplementaryMaterial online), andthe com- contained homo-polymeric repeats at numerous sites. Two
plete cDNA sequences were amplified. Each PCR run typesofmitochondrialchromosomespossessingmodule13,
consistedof35cyclesofdenaturingat94(cid:2)Cfor30s,anneal- whichwere2,682and3,185bp,werecompletelysequenced
ingat55(cid:2)Cfor30s,andextensionat72(cid:2)Cfor2min.
by primer walking and deposited in Genbank as LC114082
and LC114083, respectively. The circular architecture of the
IdentificationofModuleRegionsforEachMitochondrial
module 13 chromosome was confirmed by PCR with the
Gene
inward primer set (i.e., fig. 1B, inw. “m13 F and inw. m13
To identify the modules of each mitochondrial gene, BLAST R” in supplementary table S1, Supplementary Material
2.2.28+(ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.28/, last online). All mitochondrial chromosomes sequenced herein
accessed9April2015)wasutilized. A local BLAST database contained nearly identical 500-bp sequences at the 50 up-
was constructed from the provided CCS reads. Module stream region of each module; these sequences were
2872 GenomeBiol.Evol.8(9):2870–2878. doi:10.1093/gbe/evw207 AdvanceAccesspublicationAugust25,2016
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Hyper-EccentricStructuralGenesintheMitochondrialGenomeoftheAlgalParasiteHemistasiaphaeocysticola
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FIG.1.—Summaryofpreliminarysurveysofcox1-codingchromosomes.(A)CompletecDNAsequenceofmaturecox1mRNA.Theannealingpositions em
oftheprimersandthemoduleregionsidentifiedinthepreliminarysurveysareshownwiththecDNAsequence.(B)Schematicrepresentationofcircular ic
.o
chromosomespossessingmodules7,12,and13.Solidlinescorrespondtotheregionsthatwereactuallysequenced.Theregionsshownbydashed-linesstill u
p
remaintobesequenced.Onthephysicalmapofthechromosomespossessingmodule13,thepositionsofprobes(i.e.,M13-1andM13-2)thatcan .c
o
distinguisheachchromosomearealsoshown.SR,sharedregion;m7,module7;m12,module12;m13,module13.(C)Southernhybridizationresults.Left, m
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middle,andrightcolumnscorrespondtotheresultsobtainedusingthespecificprobesforthesharedregion,M13-1,andM13-2,respectively. b
e
/a
rtic
designated“sharedregions(SRs)”(fig.1B).Southernhybrid- request). Homo-polymeric repeats were also recognized in le
-a
izationanalysisusingthesharedregions(withoutanyrestric- eachCCSreadatnumeroussites. bs
tion enzyme digestion) showed multiple signals (fig. 1C). tra
c
While the band patterns and intensities of the M13-1 and t/8
M13-2 probe signals were different from one another, cox1GeneStructureandSequenceVariations /9/2
8
those signals corresponded to the portion of the signals of Seventeen fragments corresponding to most of the protein- 70
/2
sharedregionprobe(fig.1C). codingregionofcox1wereretrievedfromtheCCSdata,but 6
6
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seven small regions were missing (fig. 2A). Including these 8
5
regions, the H. phaeocysticola cox1 protein-coding region 0 b
AmplificationandSequencingofMitochondrialDNA y
was found to be fragmented into at least 24 modules g
u
SequencesamplifiedbyPCRwiththeoutwardprimersofthe (fig.2A).Theboundarypositionbetweenthemodulescould e
s
sharedregion(i.e.,“CONS.fRandCONS.rF1”and“CONS. be elucidated clearly, except in a few exceptional cases. For t o
n
fRandCONS.rF2”insupplementarytableS1,Supplementary example, as the boundary between modules 7 and 8 over- 2
8
Material online) included partial sequences of cox1, cox2, lappedbyoneTresidue,nopreciseboundarywasrecogniz- M
a
cox3, cob, atp6, and nad7 (data not shown). PCR products able (fig. 2B). Five sites for U-insertion type of RNA editing rc
h
obtained using the shared region primers were divided into were predicted (i.e., between modules 11 and 12, modules 20
1
twofractionsonthebasisoflength,forexample,1-2and2- 12and13,modules19and20,andmodules20and21,and 9
20kb,togeneratetwodifferentDNAlibrariestoobtaincom- attheendofmodule24:fig.2A,CandD).Theexactnumber
prehensivesequencedata(seebelow). ofUresiduesinsertedbetweenmodules12and13aswellas
Thelibraryconstructedfrom1-to2-kbPCRproductswith modules19and20couldnotbeelucidated,becauseitwas
sharedregionprimerswassequencedbyoneSMRTcell,and unclear whether the last T residues on modules 12 and 19
227,054subreadsyielding12,992CCSreadswereobtained. wereindeedpartofmodulesornot(fig.2CandD).ThefiveT
The second library consisting of 2-20-kb PCR products was residues between modules 12 and 13 in H. phaeocysticola
sequenced by two SMRT cells, and 127,303 and 157,557 were in the same position as an RNA editing site of
subreads, yielding 9,581 and 11,542 CCS reads were ob- Diplonemapapillatumcox1,inwhichsixuridinesareinserted
tained, respectively. The raw sequence data were deposited (fig. 2A). The eight boundaries between D. papillatum cox1
inGenbankasDRA003989.TheCCSreadswereanalyzedin moduleswereexactlysharedwithcox1ofH.phaeocysticola
the subsequent analyses (CCS reads are provided upon (fig.2A).
GenomeBiol.Evol.8(9):2870–2878. doi:10.1093/gbe/evw207 AdvanceAccesspublicationAugust25,2016 2873
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Yabukietal.
A U x8 U x5? “m16” m19 U x4?U x11 m21U x3?
“m10” m15 “m18” “m22”
Hp “m4” “m6”
cox1 m1m3 m5 m7 m8 m9 m12m13m14 m17 m20 m23m24AA・・AA
“m2” m11
(LC114081)
Dp
cox1 m1 m2 m3 m4 m5 m6 m7 m8 m9 AA・・AA
U x6
B
* *
cDNA(LC114081) GTTAAGTGTCGATTGCTTCATCATAGCGTTACATGCTGTAGGTCTATCATC D
o
cDNA(LC120367) GTTAAGTGTCGATTGCTTCATCATAGCGTTACATGCTGTAGGTCTATCATC w
cDNA(LC120370) GTTAAGTGTCGATTGCTTCATCATAGCGCTACATGCAGTAGGTCTATCATC nlo
cDNA(LC120379) GTTAAGTGTCGATTGCTTCATCATAGCGTTACATGCTGTAGGTCTATCATC a
d
cDNA(LC120382) GTTAAGTGTCGATTACTTCATCATAGCGTTACATGCTGTAGGTCTATCATC e
cDDNNAA (mL7C120383) GGTTTTAAAAGGTTGGTTCCGGAATTTTGGCCTTTTCCAATTCCAATTAgAaCtGgTtTcAcCaAtTaGtCaTtGcTcAaGtGaTtCaTtAgTgCaAcTaCt d fro
DNA m8-1 ctacctcaatgcccttatgccagTAGCGTTACATGCTGTAGGTCTATCATC m
DNA m8-2 gtgctctgacgtcctatccacagTAGCGCTACATGCAGTAGGTCTATCATC h
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* * * a
cDNA(LC114081) CGTCCTACAGCATAGTGGCAATTTTTGCTTGTTCAACGTCCTCGGCATGGT de
cDNA(LC120367) CGTCCTACAACACAGTGGCAATTTTTGCTTGTTCAACGTCCTCGGCATGGT m
cDNA(LC120369) CGTCCTACAGCATAGTGGCAATTTTTGCTTGCTCAACGTCCTCGGCATGGT ic
cDNA(LC120370) CGTCCTACAACACAGTGGCAATTTTTGTCTGTTCAACGTCCTCGGCATGGT .ou
ccDDNNAA((LLCC112200337712)) CCGGCTCCCCTTAACCAAAGCCAACTAAGGTTGGGGCTAAAATTTTTTTTTTGGCCTTTTGGTTTTCCAAAACCGGTTCCCCTTCCGGGGCCAATTGGGGTT p.co
cDNA(LC120386) CGTCCTACAACACAGTGGCAATTTTTGCTTGTTCAACGTCCTCGGCATGGT m
DNA m12-1 CGTCCTACAGCATAGTGGCAATatcggcatatagcatatatgtcgatattg /gb
DNA m12-2 CGTCCTACAACACAGTGGCAATattgaggtatagcatatacctcgatattg e
DDNNAA mm1133--12 gtaatgatggggctagggttaatcggaaggggttcctcgtagtagttgttgtGGTCCTTTGGTTTTCCAAAACCGGTTCCCCTTCCGGGGCCAATTGGGGTT /artic
le
-a
b
DccDDNNAA((LLCC111240038716)) TTCCCCAAAACCGGGGAATTTTCCTTAATTGGTTTTAACCGGAATTTTTTTTGGCCGG*TCCCAATTACCCTTCTCCTTGGAGTTAACCTTAAGGGGCCAACC stract/8
cDNA(LC120378) TCCAACGGATTCTATGTTACGATTTTGCGTCATCCTTCTGGTACTAGGCAC /9/2
DNA m19 TCCAACGGATTCTATGTTACGATgtacctctacccccccccccagccggct 8
DNA m20-1 tgctaccccgcaatgtcacccccgtaGCGTCATACTCCTGATACTAGGCAC 70
DNA m20-2 atgctaccccgtgatgtcaccccgtaGCGTCACACTGCTGATACTAGGCAC /2
6
DNA m20-3 atgctaccccgtcatgccattgagtaGCGTCATCCTTCTGGTACTAGGCAC 6
9
8
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FIG.2.—SummaryofgenefragmentationpatternofHemistasiaphaeocysticolacox1.(A)Fragmentationpatternofcox1ofHemistasiaphaeocysticola 0 b
(Hp)andcomparisonwiththatofDiplonemapapillatum(Dp).TheRNAeditingsitesatwhichuridineresiduesareinsertedareshownbyblackverticalbarsin y g
thesequence(e.g.,betweenm11andm12),andthenumberoftheinserteduridinesisindicatedwiththearrowateachsite.Regionsthatstillremaintobe ue
s
detectedinthemitochondrialDNAsequencesareshownasthedashed-lineboxes,andtheirtentativemodulenumbersareshownindoublequotation t o
n
marks.Verticaldouble-headedarrowsshowtheboundarypositionssharedbetweenH.phaeocysticolaandD.papillatum.(B–D)Junctionregionsofmodules 2
7and8,modules12and13,andmodules19and20.Modulesequencesingenomicdataareshowninbold.Theoverlappingresidueisshowninwhitetext 8 M
onblackbackground,andpossiblesitesforU-insertionaremarkedbyblackdots.Thesitesatwhichthenucleotidepolymorphismcanberecognizedare a
rc
highlightedingray.ThesitesatwhichthenucleotidepolymorphismisrecognizedinonlycDNAsequencesareadditionallymarkedbysingleasterisks.The h
2
accessionnumbersofcDNAsequencesareshowninbracketsandthenumbersshownwiththemitochondrialDNAsequences(e.g.,m8-1andm8-2) 0
1
correspondtothesequencenumbersprovidedinthesupplementarymaterial,SupplementaryMaterialonline. 9
Among the 17 identified modules in H. phaeocysticola werealsorecognizedinthecDNAsequencesamplifiedwith
cox1, sequence variation was found in 12 modules. Among theprimerset,“cox1FULLF”and“cox1FULLR.”Sequencing
thevariations,therewerealsononsynonymoussubstitutions. of 34 clones yielded 25 different sequences (deposited in
Forexample,fourdifferentvariantsofmodule20wereiden- Genbank as LC114084–LC114087 and LC120367–
tified in the mitochondrial DNA sequences (fig. 2E, supple- LC120387). Although eight cDNA sequences could not be
mentary material, Supplementary Material online), three of translated to amino acid sequences (see below), the other
whicharenonsynonymoussubstitutions.Theselectedrepre- sequences were also different from one another at the
sentative sequences of each module in which the variations amino acid level (supplementary material, Supplementary
were found are provided in the supplementary material, Materialonline).Furthermore, severalsinglenucleotidepoly-
Supplementary Material online. The sequence variations morphismsfoundincDNAsequencescouldnotbeconfirmed
2874 GenomeBiol.Evol.8(9):2870–2878. doi:10.1093/gbe/evw207 AdvanceAccesspublicationAugust25,2016
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Hyper-EccentricStructuralGenesintheMitochondrialGenomeoftheAlgalParasiteHemistasiaphaeocysticola
A C
** ** ** *** **** * * **** truncated “m2” “m22”
cDNA(LC114081) ATGTTGGTGGTTTTAGGTTTTTTTTGAACGTGCAACGCTAAGGTCATTGGATTCGTATATTTAG
cDNA(LC120367) GGGTTTTAGGTTTTTTTTGAACGTGCAACGCTAAGGTCATTGGATTCGTATATTTAG
cDNA(LC120368) TAGTTTTAGGTTTTTTTTGAACGTGCAACGCTAAGGTCATTGGATTCGTATATTTAG LC114084 m1 m23m24
cDNA(LC120382) p r i m e r TGGTTTTGGGTTTTTTTTAAACGTGCAACGCTAAGGTCATTGGATTCGTATATTTAG
cDNA(LC120383) (c ox 1 FU LL F )TGGTTTTGGGTTTTTTTTGAACGTGCAACGCTAAGGTCATTGGATTCATATATTTGG
cDNA(LC120384) TGGTTTTGGGTTTTTTTTGAACGTGCAACGCTAAGGTCATTGGATTCGTATATTTGG
cDNA(LC120386) TGGTTTTGGGTTTTTTTTGAACGTGCAACGCTAAGGTCATTGGATTCGTATATTTAG GTATATTTAGTTTTTGGTGTGGTGTTGGTGGGGTTGGTCCTT
DNA m1-1 ATGTCAATAGCCTCAGGTCCTCTTTGAACGTGCAACGCTAAGGTCATTGGATTCGTATATCTAG
DNA m1-2 ATGTCAATAGCCTCAGGTCCCTTTTGAACGTGCAACGCTAAGGTCATTGGATTCGTATATCTAG m1 truncated “m22” m23
DNA m1-3 ATGTTAGTAGTCCCAGGTCCCTTTTGAACGTGCAACGCTAAGGTCATTGGATTCGTATATCCAG “m2”
B D
truncated of “m2” truncated “m18”
cDNmAis ascincge sresgioionn nsu amnbde rs sequences T & G / N (%) LC114085 m1 mm1290 m23m24
“m2” LLLCCC111112440003886167 TCTTTTTTTTTTTTTGGGGGGTTTGGGTTTGGGGGGTGGGGGTTTTTTTTT 111767///111777 (((19100400.1%%%))) m21“m22” Dow
LC114084 TTTTTGG---------- 7/7 (100%) n
GTATATTTAGTTTTTGGCTGGTGTTTTGGTTGTTTTGTTTTTGTTGATAGGTG lo
“m4” LLLCCC111122400033868185 CCCTTTGGGCCCTTTGGGGGGGGGTTTGGGAAACCCAAACCCGGGCCCAAAGGGTTTTTTCCCTTTAAATCCAAAAAACCCGCGTTTGGGTTTTTTAAAAAATTTCCCAAACCCCCCAAAGGGTTT 222120///444222 (((554027%..46%%))) m1 tru“nmc2a”ted truncated “m18” m19 aded
LC114081 TTGGTTGCTACCACTAGGGTTTTTGTTGTTGGTAGGGAGCGGGGTATTTGATGAGG 43/56 (76.8%) E fro
LC120368 TTGGTTGCTACCACTAGGGTTTTTGTTGTTAGTAGGGAGCGGGGTATTTGATGAGG 42/56 (75%) m
“m6” LLLLCCCC111122220000333367789150 TTTTTTTTGGGGGGGGTTTTTTTTGGGGCCCCTTTTAAAACCCCCCCCAAAACCCCTCTTAAAAGGGGGGGGGGGGTTTTTTTTTTTTTTTTTTTTGGGGTTTTTTTTGGGGTTTTTTTTGGGAGGGGTTTCGAAAGGGGGGGGGGGGAAAAGGGGCCCCGGGGGGGGGGGGGGGGTTTTAAAATTTTTTTTTTTTGGGGGAAATTTTGGGGAAAAGGGGGGGG 44442342////55556666 ((((77775685%..%88%%)))) LC1140“8m62”m1 mmtr12u90ncatemd2 “3mm1284” https
LC120384 TTGGTTGCTACCACTAGGGTTTTTGTTGTTGGTAGGGAGCGGGGTACTTGATGAGG 42/56 (75%) m21“m22” ://a
LC114081 GTTTTTTGACTGCGCGTCC 13/19 (68.4%) ca
“m10”LC120373 GTTTTTCGACTGCGCGTCC 12/19 (63.2%) TATTTAGCTTTTGGTGTGGTGTTTGGGGTTTTGGTTGTGTTGTTTTTATTGATAG d
LC120379 GTTTTTTGATTGCGCGTCC 14/19 (73.7%) m1 “m2” truncated “m18” m19 em
“m16”LC114081 TTTTTGTGGGTTTGGGTTTTT 21/21 (100%) ic
.o
LC114081 GGGAGCAGTTTTTGGTGTTTTGGTTGGTGTTTTGGTTGTTTTGTTTTTG 46/49 (93.9%) u
“m18”LLLCCC111222000333667783 GAAGGGGGGAAAGGGCCCAAAGGGTTTTGGTTTTTTTTTGGGGGGTTTGGGTTTTTTTTTTTTGGGGGGTTTTTTGGGGGGTGGGGGTTTTTTTTTTTTGGGGGGTTTTTTGGGTTTTGGTTTTTTGGGTTTTTTTTTTTTTTTGAA 444554///444999 (((998119...888%%%))) F LC114087m1m3“?” m5 m7 m8 p.com
LLLCCC111211044300888475 GA-GG-GG-AA-GG-CC-AA-GG-TT-TG-TT-TT-TT-GG--GAT-TG-G-TT-TTT-TT-TG-G-GGTCTTTCGGGGGGTGTGGGTTTTTTTTTTTTGGGGGGTTTTTTGGGTTTTGTTTTTTTGGGTTTTTTTTTTTTTTTGGG 442545///442996 (((989196...882%%%))) “m2” m“m116” “m16” m19 “m22” /gbe
LC114086 -------------------------GGGGTTTTGGTTGTGTTGTTTTTA 23/24 (95.8%) m9 m12m13m14 m17 m20 m23m24 /a
“m22”LC114081 TGTGGTGTTGGTG 13/13 (100%) “m10” m15 “m18” m21 rtic
le
-a
FIG.3.—(A)MultiplealignmentofcDNAandgenomicsequencesofmodule1ofcox1.Thesitesatwhichthenucleotidepolymorphismcanbe bs
recognizedarehighlightedingray.ThesitesatwhichthenucleotidepolymorphismisrecognizedononlycDNAsequencesaremarkedbysingleasterisks,and tra
c
thesitesatwhichallnucleotidesareinconsistentbetweencDNAandgenomicsequencesaremarkedbydoubleasterisks.(B)Multiplealignmentsofthe t/8
cDNAsequencesoftheregionsthatstillremaintobedetectedinthemitochondrialDNAsequences.G/Tresiduesareshowninwhitetextonblack /9
/2
background.TheG/Tcontentandpercentageofeachsequencearealsoshownintherightcolumn.(C–E)cDNAsequencesofpossibleimmaturecox1mRNA 8
7
0
lackingtheinternalregion.(F)cDNAsequenceofpossiblemisassembledcox1mRNA,inwhichmodule4isreplacedwithanunknownsequence. /2
6
6
9
8
in these genomic sequences (figs. 2 and 3A) and all cDNA sequence(fig.3F).AstopcodonwasfoundinsideLC120371. 5
0
sequencescorrespondingtothemodule1regionwereincon- ThethreeothercDNAsequences(i.e.,LC120375,LC120380 b
y
sistentwiththegenomicsequencesatseveralsites(fig.3A).At andLC120382)lackedoradditionallypossessedoneTresidue g
u
e
these inconsistent sites, G and T in cDNA sequences were incontinuousTregions. s
t o
replacedwithAandCinthegenomicsequences,respectively. n
2
Seven unidentified regions in cDNA sequence were rela- 8
GeneStructuresofcob,cox2,andnad7 M
tivelyT/G-rich(fig.3B).Inparticular,theT/Gcontentof“mod- a
ules 2, 16, 18, and 22” exceeded 90% (fig. 3B). Sequence ThecompletecDNAsequencesofmaturecob,cox2,andnad7 rch
variationswerealsorecognizedincDNAdataforthesemiss- mRNAweredeterminedbyRT-PCRand30/50 RACEmethods, 20
1
ing regions, except “modules 16 and 22” (fig. 3B). Among andtheirsequencesaredepositedinGenbankasLC114088, 9
eightcDNAsequencesthatcouldnotbetranslatedtoamino LC114089, and LC114090, respectively (fig. 4). These se-
acid sequences, internal region was missing in three se- quencescouldalsobetranslatedtoaminoacidsequencesby
quences (i.e., LC114084–LC114086: fig. 3C, D and E); for geneticcodetable4.Mostofthemodulesofeachgenewere
example,the30 endoftruncatedmodule2wasdirectlycon- identifiedinthemitochondrialDNAsequencesandthemissing
nectedwiththe50 endofmodule22inLC114084(fig.3C). regionsexhibitedhighG/Tcontentsaswasthecaseforcox1
While both LC114085 and LC114086 contained truncated (fig. 4). Each gene was fragmented more finely than in
module 18, the length of and truncated position in module D. papillatum, and the boundaries between D. papillatum
18differed(fig.3DandE).Inonesequence(i.e.,LC114087), modulesweremostlysharedinH.phaeocysticola,exceptone
only module 4 was replaced with the unknown sequence, position in each gene (e.g., the boundary position between
which could not be identified in the mitochondrial DNA modules5and6incobofH.phaeocysticola;fig.4).RNAedit-
data, and the codon-frame was shifted from this unknown ingsitesforU-insertionwerealsofoundincob,cox2,andnad7
GenomeBiol.Evol.8(9):2870–2878. doi:10.1093/gbe/evw207 AdvanceAccesspublicationAugust25,2016 2875
GBE
Yabukietal.
shifted by 8 bp
U x1 U x5 55/57 89/145 29/33
Hp m5 “m9” m13“m14”
cob m1 m2 m3 m6 m7 m8 “m11” m12 m15 AA・・AA
(LC114088) “m4” m10
46/65
Dp
cob m1 m2 m3 m4 m5 m6 AA・・AA
U x3
shifted by 2 bp (at least)
10/10 U x4 U x4 U x13 D
o
coHxp2 m1 “mm34” m5 m6 m7 m8m9m10 AA・・AA wnlo
m2 a
(LC114089) d
e
d
Dp fro
cox2 m1 m2 m3 m4 AA・・AA m
h
7/12 7/7 3/3 11/“1m212” U x3 7/8 shifted by 3 bp U x6 ttps://a
Hp 48/93 “m5” “m7” “m9” m13 “m18” cad
nad7 “m1” m6 m8 m11 m14 m15 m16m17 m19 m20 m21 m22 AA・・AA em
(LC114090) m“m23” m4 m10 ic.o
5/9 u
p
Dp .c
nad7 m1 m2 m3 m4 m5 m6 m7 m8 m9 AA・・AA om
/g
b
e
FIG.4.—Fragmentationpatternsofcob,cox2,andnad7ofHemistasiaphaeocysticola(Hp)andcomparisonwiththoseofDiplonemapapillatum(Dp). /a
RNAeditingsitesatwhichuridineresiduesareinsertedareshownbyblackverticalbars,andthenumberofinserteduridineresiduesisindicatedwiththe rtic
le
arrowateachsite.Missingregionsareshownasdashed-lineboxes,andtheirtentativemodulenumbersareshownindoublequotationmarks.Vertical -a
b
double-headedarrowswithsolidlinesshowtheboundarypositionssharedbetweenH.phaeocysticolaandD.papillatum.TheG/Tcontentsofeachmissing s
regionareshownintheboxes. tra
c
t/8
/9
/2
asincox1.TheRNAeditingsitefoundattheterminalregionof 2011), no other multi-copy modules have been reported in 8
7
0
cobinD.papillatumwasnotfoundinH.phaeocysticola(fig.4). other diplonemids. In addition, sequence variation within a /2
6
Thegenefragmentationpatternsaswellasthepositionand given module has not been reported in other diplonemids. 6
9
8
numberofUresiduesinsertedbyRNAeditingaresummarized Furthermore, the mitochondrial chromosomes of H. phaeo- 5
0
in figure 4; it should be noted that this schematic summary cysticola are smaller than those of other diplonemids, b
y
mightnotbepreciselycorrectbecauseofoverlappingneighbor although only two chromosomes (i.e., LC114082 and g
u
e
module sequences at several boundary positions (e.g., be- LC114083) were sequenced completely in the present s
t o
tweenmodules7and8ofcob;seesupplementarymaterial, study. Other chromosomes of H. phaeocysticola amplified n
2
Supplementary Material online). Sequence variations in the by module-specific outward primers and incompletely se- 8
M
moduleswerealsorecognizedbycomparisonwiththegeno- quenced were also shown to be smaller than 5 kb (fig. 1B). a
rc
micsequencesofthethreegenes,aswithcox1,andtheirse- Other chromosomal features—the existence of a shared h
2
lected representative sequences are also provided in the region and many homo-polymeric repeats—are shared with 0
1
supplementarymaterial,SupplementaryMaterialonline. otherdiplonemids. 9
As the existence of such a shared region has also been
reported in the chromosomes of other diplonemids (Vlcek
Discussion
etal.2011;Kiethegaetal.2013),thisisnotauniquefeature
The four genes examined here are fragmented into small of H. phaeocysticola. However, this feature is useful for se-
pieces in the mitochondrial genome of H. phaeocysticola, quencingthemitochondrialgenomeofH.phaeocysticola,be-
and no nonfragmented versions of the genes were found. cause comprehensive mitochondrial genome amplification
Thesefeaturesareconsistentwiththoseofotherdiplonemids. can be expected by PCR with outward primers annealing
Furthermore, many modules had multiple copies whose se- within the shared region. In fact, the modules of other
quencesdifferedfromoneanother,eventhoughtheycorre- genes (i.e., cob, cox2, nad7, and more) were recovered
spondtothesamecodingregion.Althoughmodule9ofcox1 from the amplified sequences in the preliminary analyses.
ofD.papillatumwasfoundintwochromosomes(Vlceketal. Thus, this approach allowed us to selectively amplify the
2876 GenomeBiol.Evol.8(9):2870–2878. doi:10.1093/gbe/evw207 AdvanceAccesspublicationAugust25,2016
GBE
Hyper-EccentricStructuralGenesintheMitochondrialGenomeoftheAlgalParasiteHemistasiaphaeocysticola
Complexity of Mt gene structure possibly reached to the plateau
and did not increase so much from this point.
Diplonema D
Rhynchopus i
C p
l
The evolutionary constraint on o
n
the complex gene structure
e
B′ changed at this branching point. m
e D
E a ow
B n
lo
a
d
e
Hemistasia d
fro
Further fragmented genes were established. m
A (e.g., 24 pieces of cox1, 10 pieces of cox2) http
s
D Gene fragmentation continued and RNA editing sites increased. ://a
c
a
d
Kinetoplastids em
ic
B′ .o
u
Euglenids p
.c
o
m
FIG.5.—SchematicsummaryoftheevolutionaryhistoryleadingtotheeccentricstructuralgenesfoundinHemistasiaphaeocysticolamitochondriabased /gb
onthefindingspresentedinthisstudyandpreviousstudies(Flegontovetal.2011;Doba´kova´ etal.2015;YabukiandTame2015).(A)Emergenceofthe e/a
fragmented genes in the mitochondrial genome. (B) Emergence of U-insertion and C-to-U types of RNA editing. This evolutionary event may have rtic
independentlyoccurredinthelineagesleadingtokinetoplastidsanddiplonemids,respectively(shownasB0).(C)EmergencesofA-to-Isubstitutiontype le
-a
ofRNAediting.MostofgenestructuresfoundinDiplonemaandRhynchopuswereestablishedinthecommonancestorofalldiplonemids.(D)Emergenceof b
s
U-deletiontypeofRNAediting.(E)EmergenceofthesecondaryRNAinsertionintothepre-assembledRNA. tra
c
t/8
/9
/2
8
7
0
mitochondrial genome of H. phaeocysticola and sequence it Seven small fragments of cox1 could not be identified in /2
6
broadlyanddeeplyusingPacBioRSII(PacificBiosciences). themitochondrialDNAsequences(fig.2A).Althoughthepos- 6
9
8
Module identification based on a BLAST search showed sibilitythatsomechromosomeswerenotamplifiedwiththe 5
0
thatcox1ofH.phaeocysticolaisfragmentedintoatleast24 PCRconditionsusedinthepresentstudyand/orthatthese- b
y
pieces(fig.2A).AsthreeothergenesofH.phaeocysticolaare quencingdepthwasinsufficientcannotberuledoutcomple- g
u
e
also fragmented >two times finer than the corresponding tely,weareinclinedtoconsiderthattheymaynotbeencoded s
t o
genes in D. papillatum, this hyper-fragmentation is possibly directlyinthegenomeand/ormaybefurtherfragmentedinto n
2
ageneralfeatureofthemitochondrialgenesofH.phaeocys- muchsmallerpieces.Theseunidentifiedregionsarepredom- 8
M
ticola.Thegenesfoundinthemicronucleargenomesofthe inantlycomposedofTandGresidues(fig.3B)andseveralC a
rc
hypotrichous ciliates are also discontinuous and highly inter- andAresiduesinthegenomicsequencesarereplacedwithT h
2
rupted,butthesearejoinedbyDNAsplicingduringmacronu- and G residues in cDNA sequences, respectively (fig. 3A). If 0
1
cleardevelopmentandonlythenoninterruptedgenesofthe theseinconsistenciesbetweencDNAandgenomicsequences 9
macronuleargenome aretranscriptionallyactive (Landweber representtheexistenceofC-to-UandA-to-G(orI,becauseI’s
et al. 2000; Swart et al. 2013). Hence, the mitochondrial inRNAarereadasG’sbyreversetranscriptase)typesofRNA
genesfoundinH.phaeocysticolamaybethemoststructurally editing,thegenomicsequencescorrespondingtosomeofthe
eccentric transcriptionally active genes known to date. unidentified regions are different from the cDNA sequences
Furthermore, U-insertion RNA editing in the mitochondrial and therefore they still remain to be identified. Indeed, the
transcripts of H. phaeocysticola was also predicted to occur existenceofC-to-UandA-to-ItypesofRNAeditinghasbeen
more frequently compared with homologs in other diplone- confirmed in D. papillatum recently (Moreira et al. 2016).
mids:forexample,H.phaeocysticolahasmorethantwoRNA Hence,itisreasonabletoconsiderthatthesameRNAediting
editingsitesonanaveragepermitochondrialgene,whereas mayalsooccurinthemitochondrionofH.phaeocysticola.
one editing site wasfound incox1, cox2andcobandnone Furthermore, the cox1 cDNA sequences that possess the
wasfoundinnad7inD.paillatum(Moreiraetal.2016). truncatedmodule sequences(fig. 3C, D and E) suggest that
GenomeBiol.Evol.8(9):2870–2878. doi:10.1093/gbe/evw207 AdvanceAccesspublicationAugust25,2016 2877
GBE
Yabukietal.
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ThisworkwassupportedbygrantsfromtheJSPSawardedto
A.Y.(26840133)andK.T.(25650132). Associateeditor:SandraBaldauf
2878 GenomeBiol.Evol.8(9):2870–2878. doi:10.1093/gbe/evw207 AdvanceAccesspublicationAugust25,2016