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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 • Chromosomehomologuescanvarysignificantlyinsize • 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 • Gomezetal.(2010)RegulationofGeneExpressioninProtozoaParasites.JournalofBiomedicineandBiotechnology.http:// dx.doi.org/10.1155/2010/726045 • Clayton(2014)NetworksofgeneexpressionregulationinTrypanosomabrucei.MolecularandBiochemicalParasitology. 195(2):96-106.http://dx.doi.org/10.1016/j.molbiopara.2014.06.005 • Forrester&Hall(2014)Therevolutionofwholegenomesequencingtostudyparasites,MolecularandBiochemical Parasitology.195,(2):77-81.http://dx.doi.org/10.1016/j.molbiopara.2014.07.008 • El-Sayedetal(2005)ComparativeGenomicsofTrypanosomatidParasiticProtozoa.Science:404-409 • Teixeiraetal(2012).Trypanosomatidcomparativegenomics:Contributionstothestudyofparasitebiologyanddifferent parasiticdiseases.GeneticsandMolecularBiology,35(1),1–17.PMC3313497 • Siegeletal(2009).FourhistonevariantsmarktheboundariesofpolycistronictranscriptionunitsinTrypanosomabrucei.Genes &Development,23(9),1063–1076.http://doi.org/10.1101/gad.1790409 • Taylor&Rudenko(2006)Switchingtrypanosomecoats:what'sinthewardrobe?TRENDSinGenetics.22(11):614-620.http:// dx.doi.org/10.1016/j.tig.2006.08.003 • Nakaaretal(1994).UpstreamtRNAgenesareessentialforexpressionofsmallnuclearandcytoplasmicRNAgenesin trypanosomes.MolecularandCellularBiology,14(10),6736–6742.PMC359204 • JessicaC.Kissinger,JeremyDeBarry(2011)Genomecartography:chartingtheapicomplexangenome,TrendsinParasitology. 27(8):345-354.http://dx.doi.org/10.1016/j.pt.2011.03.006 • Jiangetal(2013)PfSETvsmethylationofhistoneH3K36repressesvirulencegenesinPlasmodiumfalciparum.Nature.499: 223-227. • Ulluetal(2002)RNAiinterference:advancesandquestions.Phil.Trans.R.Soc.Lond.B.357:65–70.