Download Genomes and Gene Expression in Parasitic Protozoa

Genomes and Gene
Expression in Parasitic
Protozoa
Gene organisation
Genomes and Gene Expression in
Trypanosomatids
Trypanosomabrucei
Trypanosomacruzi
Leishmaniaspp
The Tritryps
• Genomesequencingprojects–mid1990’s
• L.majorandT.bruceisequencedusingpurifiedchromosomelibraries,andwhole
genomeshotgun(laterpublications)
• T.cruzisequencedbywholegenomeshotgun(hybridgenome).
El Sayed et al, 2005
The Kinetoplastid Genome
Splitover2organelles
-TheKinetoplast(kDNA)–mitochondrialsubunits(20genes)
-TheNucleus–allothergenesrequired(~10,000genes)
GC-ATbias–50%
The Mitochondrial genome
• mRNAisextensivelyedited
• DispensableforBloodstreamformT.brucei
• DNAisdividedintogRNAguidesandpre-mRNA
thatencodesformitochondrialsubunits
Chromosome organisation of T. brucei
•
•
•
•
Trypanosomesarediploid
Genometotals~40Mb(perhaploidgenome)
GenesaretranscribedbyRNAPolI,RNAPolIIandRNAPolIII
Organisedintopolycistronictranscriptionunits(directionalgeneclusters)
Akiyoshi & Gull, 2013
The T. brucei centromeres
• Regionsbetweendirectionalgeneclusters
• Containtransposableelementsandanarrayof147bptandemrepeatswhichareAT-rich
• Predictedtoextendbetween20and100kb.
Echeverry et al. 2012.
Obado et al, 2007.
Classical Gene Expression: Prokaryotes vs.
Eukaryotes
Polycistronic
Exons
Transcriptionalcontrol
Monocistronic
Introns+Exons
TranscriptionalandPost-transcriptionalcontrol
RNAmodified-5’capand3’polyadenylation
-splicing
Controls of Gene Expression
Polycistronic Transcription
ConvergentSSR
Chromosome
DNA
mRNA
DivergentSSR
Gene Expression T. brucei
• Polycistronic
• The5’endofeachmRNAisgeneratedbytransplicingofacapped“splicedleader”(SL,39nt)
• SplicingiscoupledwiththepolyadenylationofprecedingmRNA
Teixeira et al, 2012
Where does transcription start and end?
Transcription
Initiation
Termination
Siegel et al ,2009
RNA Polymerase I Transcription Units
• rRNAgenesandprotein-codinggenes–variablesurfaceglycoprotein(VSG)andprocyclin
• VSGgenesaretranscribedfromBloodstreamformExpressionsites(BES)
• LocatedinthesubtelomeresoftheMBCsandICs.
• ~60kbinlength,containingES-associatedgenes(ESAGs)andtheVSGgeneatthe3ʹend
Horn, 2014
El Sayed et al, 2005
Antigenicvariationcoveredon29thFebruary
RNA Polymerase II Transcription Units
RNA Polymerase III Transcription Units
Nakaar et al, 1994
• RNApolIIItranscribesanumberofsmallnoncodingRNAsthatactintranslationorother
cellularprocesses.
• TrypanosometRNAsarefoundwidelyinterspersedbetweenthegeneclustersonthe
megabasechromosome.
• LinkageoftRNAandsmallRNAgenesiswidespreadinthetrypanosomatids
• TheinternalcontrolregionsofthesmallRNA-associatedtRNAgenesserveasbifunctional
regulatoryelements.
GCG
GCA
GCU
GCC
UUU–0.16
UUC–0.84
CAU
CAC
AUA
AUU
AUC
His
Ile
AAA–0.04
AAG–0.96
AGU
AGC
UCG
UCA
UCU
UCC
ACG
ACA
ACU
ACC
GUG
GUA
GUU
GUC
UAU
UAC
Ser
Thr
Val
Tyr
Arg AGG
AGA
CGG
CGA
CGU
CGC
Gln CAG
CAA
Pro CCG
CCA
CCU
CCC
Asn AAU
AAC
Leu UUG
UUA
CUG
CUA
CUU
CUC
Lys AAG
AAA
GGG
GGA
GGU
GGC
Gly
Phe UUU
UUC
Glu GAG
GAA
Asp GAU
GAC
Cys UGU
UGC
Ala
Relativefrequency
T. brucei and Leishmania have codon bias
1
T.brucei
0.8
L.major
0.6
0.4
0.2
0
Preferred tRNAs –preferred codons in
DNA sequence
Horn, 2008.
Codon-bias and genome organisation
CAI
Strand-switch
Tandemduplications
VSGs
Directionoftranscription
T.brucei
Chr.3
L.major
Chr.1
CAI
Strand-switch
Tandemduplications
Directionoftranscription
Geneswithlowbias(includingVSGs)tendtoassociatewith(divergent)strand-switches
Tandemduplicationstendtohavehigherbias
Unusual aspects of kinetoplastid gene
organisation and expression
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Chromosomehomologuescanvarysignificantlyinsize
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T.bruceialsocontainstwoclassesofatypicalnuclearchromosomes;
- IntermediatechromosomesthatcontainsomeVSGgenes(nohouse-keepinggenes)
- Minichromosomes(reservoirofVSGsequences).
•
Transcriptionispolycistronic,andprocessinginvolvesatrans-splicingmechanisminwhichall
mRNAsaremodifiedpost-transcriptionallybytheadditionofa39-nucleotidesplicedleader
totheir5'-ends.
•
ProteincodinggeneslackconventionalRNApolymeraseII(polII)promotersandare
organisedinlongco-directionalclusterswhichcanstretchfortenstohundredsofkilobases.
Genomes and Gene Expression in Apicomplexans
Plasmodiumfalciparum
Toxoplasmagondii
Cryptosporidium
The Apicomplexans
• Genomesequencingprojects(several)–startedmid-1990’s
• Wholechromosomelibrariesandshotgunapproach
Kissinger & DeBarry, 2011
The Malarial Genome
Splitover3organelles
-TheApicoplast–35kb–encodes30proteins
-TheNucleus–allothergenesrequired(~5,500genes)
-TheMitochondria
ATbias–80%-risingto90%inintergenicregions
Chromosome organisation of P. falciparum
• InthemammalianHostP.
falciparumisHaploid
• Genometotals~40Mb
• GenesaretranscribedbyRNA
PolIIonly
• Subtelomeresarealsohighly
variable(multigenesforimmune
evasion).
Antigenicvariationcoveredon22rdMarch
Gardner et al, 2002
Gene Expression P. falciparum
• Generallymonocistronic,butonebicistronicunit(erythrocytebindingprotein+ATPsynthase)
• PolIItranscription
• Reliesonspecificinteractionwithtransactingfactorswithsequencesupstreamofthe
transcriptionstartsites
Histone modifications also responsible for var
gene silencing
• KnockdownofpfSET2vs(methyltransferase)
causestheexpressionofmultiplevargenes
• Methylationofhistonesassociatedwith
repressedvargenes
Jiang et al ,2013
Unusual aspects of Plasmodium gene
organisation and expression
AlthoughverymuchlikeclassicEukaryotegeneexpression
• Flexibilityintranscriptionstartsites(expansionofTATAbox-likesequences)
• Inintra-erythrocyticstage–thepre-initiationcomplexisassembledonthepromotersofall
intra-erythrocytic-expressedgenes(independentoftranscriptionalactivity)
López-Estrano et al, 2007
The Protozoal Genetic toolbox
Or,howtomanipulategeneexpression…..
RNAi – making use of the defence machinery
Low/Highthroughput
Balaña-Fouce & Rafael, 2007
Trypanosomabrucei
Toxoplasmagondii
Cryptosporidium
Leishmaniabrasiliensis
Plasmodiumfalciparum
Leishmaniamajor
Trypanosomacruzi
Glover et al, 2015
Gene knock-out – remove a functioning gene
Plasmids/PCR/Cas9/Cre-Lox
Carnes et al, 2005
Gene over-expression
Low/Highthroughput
Carnes et al, 2005
Begolo et al, 2014
References
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