1820Regulatory RNAs and Genome AnalysisRNA调控和基因组分析.docx

1820Regulatory RNAs and Genome AnalysisRNA调控和基因组分析.docx

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1820RegulatoryRNAsandGenomeAnalysisRNA调控和基因组分析

RegulatoryRNAs

Chapter18

I.RegulatoryRNAs

A.RegulationbyRNAsinBacteria

B.RNAInterferenceinEukaryotes

C.SynthesisandFunctionofmiRNAs

D.RNAiasaToolforRegulatingGeneExpression

E.RegulatoryRNAsandX-Inactivation

F.RNAiandHumanDisease

G.Post-transcriptionalRegulation

H.FeedbackInhibition

II.GenomeAnalysis

A.DNAmicroarrays

B.IdentificationofRegulatoryDNASequences

III.SystemsBiolog

A.RegulationbyRNAsinBacteria

FormanyyearsscientistshaveknownthatsmallRNAmoleculesregulategeneexpressioninbacteria.

SomesmallRNAmoleculesregulatetranscription.

Forexample,the6SRNAinE.colibindstotheσ70subunitofRNApolymerase.

Whatroledoessigmaplayintranscription?

σ（sigma）actsasaspecificityfactor.ItspecificallybindstopromotersequencesintheDNA.

The6SRNAbindstoσ70andreducestranscriptionatmanyofthepromotersthatσ70bindsto.Therearelargeramountsofthe6SRNAduringthestationaryphaseofcellgrowth.

Thereare3phasesinbacterialcellgrowth:

1.lagphase

2.exponentialphase

3.stationaryphase

Activelydividingbacteriaareintheexponentialgrowthphase.Afterthenumberofcellsintheculturegetstoohigh,thegrowthslowsandcellsenterthestationaryphase.Bacteriathataretakenfromthestationaryphaseandplacedinfreshmediawillgrowquicklyagain,butonlyafteraninitiallagphase.

Thecellsmakemore6SRNAduringthestationaryphaseofgrowth.DuringthestationaryphaseadifferentσfactorisproducedcalledσS.σSbindstodifferentpromotersthanσ70.Thesepromotersareingenesthathelpthecellssurvivethestressofthestationaryphase.

The2σfactorscompetewitheachothertobindtothecoreRNApolymerase.

Thus,the6SRNAhelpsthecellstoshiftfromexpressinggeneswithpromotersboundbyσ70thatareneededduringexponentialgrowth,toexpressinggeneswithpromotersthatareboundbyσSthatareneededduringstationaryphase.

ThereisanothergroupofsmallRNAsinbacteriathatregulatetranslationandmRNAdegradation.TheyarecalledsmallRNAsorsRNAs.

Theyareabout80-110basepairslong.

E.colihasnearly100sRNAs.

About12ofthesRNAsinE.colihavebeenwellstudied.

MostbasepairwithcomplementarysequencesintargetmRNAs.ThiscausesthedegradationofthemRNA,orinhibitsitstranslation.Thoughinsomecasesitcanactuallyincreasetranslation.

BindingofasRNAinhibitstranslationbyblockingtheribosomebindingsite.

BindingofthesRNAactivatestranslationbyfreeingaribosomebindingsite.

B.RNAInterferenceinEukaryotes

InRNAinterference（RNAi）veryshortRNAsrepressexpressionofcertaingenes.

TheshortRNAscaninhibittranslation,causethedegradationofthemRNA,orrepresstranscriptionofthegene.

ScientistscanusethesesmallRNAstosilenceorturnoffexpressionofagene.ThismakestheseRNAsapowerfultoolforresearch.

SmallRNAsmadebyscientistsforgenesilencingarecalledsmallinterferingRNAsorsiRNAs.

ThesmallRNAsproducednormallybycellsarecalledmicroRNAsormiRNAs.

miRNAsareproducedfromlongerRNAmoleculesandthentheyarecutbyDicer.

DicercutsthelongermRNAsintoshortpiecesabout21-23basepairslong.

DicercutsboththenaturallymademiRNAsandalsocutsthesmallinterfering（siRNAs）thatareaddedbyscientists.

ThemiRNAsandsiRNAsinhibitexpressionofgenesin3ways:

1.TheycausedegradationofthemRNA

2.TheyinhibittranslationofthemRNA

3.Theycausemodificationsinthechromatintosilenceitstranscription.

ThemiRNAsorsiRNAsbindtoaRNA-inducedsilencingcomplex（RISC）.

TheRNAisdenatured.OnestrandiscalledtheguideRNAandtheotheristhepassengerRNA（whichisusuallydiscarded）.

TheRISCbindstotargetmRNAswiththeguideRNA.ThetargetmRNAsaredegradediftheirRNAiscloselycomplementarytotheguideRNA.Ifthesequencesarenotascomplementary,translationofthetargetmRNAwillbeinhibited.

TheRISCcanalsoenterthenucleuswhereitcanrecruitotherproteinsthatmodifythechromatinnearthepromoterofthegenethatiscomplementarytotheguideRNA.Thiswillstoporsilencetranscriptionofthegene.

UsingRNAitosilencegenesisveryefficient.OftenonlyverysmallamountsofdsRNAareenoughtocompletelystopexpressionofatargetgene.ThisispartlybecausetheRISCcandegrademanymRNAs.

C.SynthesisandFunctionofmiRNAs

miRNAshaveacharacteristicstructurethatmakesthemeasytoidentify.

FirstalongerRNA,calledapri-miRNA（priforprimary）istranscribed.Thisformsahairpinstructure.Thisiscut2times.Thefirstcutreleasesthestemloop,whichiscalledthepre-miRNA.

Structuresofsomepre-miRNAs.

Thepre-miRNAscanproduce2regulatorymiRNAs,onefromeachsideofthestemloop.

Structuresofsomepre-miRNAs.

Thepre-miRNAscancomefrombothintronsequencesandexonsequences.

Thedistinctivesecondarystructure（stemloop）ofthepre-miRNAsmakesitpossibletofindthesesequencesbyscanningthegenome.Insomecasesitisalsopossibletopredictwhichtargetgeneswillberegulatedbasedonthesequencecomplementarity.

1.Drosha

DroshaisanRnaseIIIenzymethatcutsthepri-miRNAtwicetoreleasethestemloopstructureofthepre-miRNA.

DroshabindstoDGCR8（alsocalledPasha）toformthemicroprocessorcomplex.Thisbindstothepri-miRNA.Itreleasesthestemloopwhichincludesabout65-70basepairs.Thisisthepre-miRNA.

CleavagebyDroshaoccursinthenucleus.Itcutsbothstrandsofthepri-miRNAtoproducethepre-miRNA.

Droshadoesnotrecognizeaspecificsequence,butrecognizesthestem-loopstructure.Itbindstothestemandthesingle-strandedRNAbelowthestems.

Itleavesa2basepairoverhangonthe3’endofthedouble-strandedRNA.

2.Dicer

Thepre-miRNAmovesfromthenucleustothecytoplasm.HereDicercutsthe

pre-miRNAtoproducethemiRNA,whichis21-25basepairslong.

Dicerboundtodouble-strandedRNA.

3.RISC

Thedouble-strandedmiRNAproducedbyDicerbindstotheRNA-inducedsilencingcomplex（RISC）.ThentheRNAisdenaturedtoproducetheguideRNAandreleasethepassengerstrand.

TheRISCwiththeguideRNAisnowreadytobindandcutorslicecomplementarymRNAs.

RISCisacomplexofmanyproteins.ItincludesaproteinfromtheArgonautefamilyofproteins.TheArgonauteproteinhasRNaseactivityandisoftencalledtheslicer.Thus,mRNAcuttingisoftencalled“slicing”.

ManyorganismshavemorethanonekindofArgonauteprotein.Humanshave8membersoftheArgonautefamiliyofproteins.NotallthemembersoftheArgonautefamilyhaveslicer（RNase）activity.RISC’swithnonslicingArgonautesmayinhibittranslation.

ThemechanismforhowtheRISCinhibitstranslationisnotaswellunderstood.ItispossiblethatthemRNAsaremovedtoprocessingbodies（Pbodies）inthecytoplasm.Thisinhibitstranslation.

BindingofanmiRNAtoanmRNAcanalsodegradethepolyAtailofthetargetmRNA.Thisalsoinhibitstranslation.

4.GeneSilencingthroughChromatinModification

HowmiRNAssilencegeneexpressionthroughmodifyingthechromatinhasbeenbeststudiedinSchizosaccharomycespombe（S.pombe）.Thisisafissionyeastthathasbeenextensivelystudied.

S.pombewasisolatedfromAfricanbeerin1893.ItsnamecomesfromtheSwahili（Africanlanguage）wordforbeer,whichispombe.

Itisasinglecelledeukaryote.Thecellsarerod-shaped.

InS.pombetheareasofthegenomethatarenearthecentromeresaresilenced.Thissilencingisthroughhistonemodifications.

ThecentromeresinS.pombehaveacentralpartthatisuniquesequences.Oneachsideofthistherearerepeatedsequencesthatarepresentinallcentromeres.

HistonesboundtotheDNAnearthecentromereshavelowlevelsofacetylationandmethylationofthelysineatposition9ofthetailonhistone3.ThisrepressestranscriptionofthisDNA.

RNApolymerasetranscribesbothstrandsofthecentromericrepeatsequences.Thesehybridizetoformdouble-strandedRNAs.ThesebecomepartofaRISC-likecomplexcalledRITS（RNA-inducedtranscriptionalsilencing）.

TheRNAintheRITSbindstocomplementarysequencesthatarebeingtranscribedbyRNApolymerase.ThisrecruitsSwi6andCir4tothesequenceandtheymodifythehistonetails.TheRITSalsoslicestheRNAthatisbeingtranscribedbyRNApolymerase.

D.RNAiasaToolforRegulatingGeneExpression

RNAiisextremelyusefulforstudiesinthewormC.elegans.Thewormsfeedonbacteria（E.coli）andthisworksasamethodfortransportingthedsRNAintothecellsoftheworm.

TheDNAcanbeclonedintoaplasmidvector.Thenwithtransformation,thiscanbetransferredintoE.colibacteria.The

E.colibacteriawilltranscribetheDNAtoproducethedsRNAthatcansilencegenesinC.elegansafterthewormseatthebacteria.

TherearelibrariescontainingdsRNAtosilenceanygeneintheC.elegansgenome.Theselibrariescanbescreenedtotestwormsfortheresultsofturningoffanygeneinthegenome.

E.colicellsproducethedsRNAsthatwillsilencegenesintheworms.

Scientistshavedesignedandsynthesizedoligonucleotidesthatcanbeclonedintoplasmids.Whentheshortgeneistranscribed,itwillfoldintoastemloopstructurethatwillberecognizedandcutbydicer.ThesearecalledshorthairpinRNAs（shRNA）.

UsingshRNAsanygeneinthegenomecanbetargetedandsilenced.

AlibraryofgenescanbetestedwithplasmidsthateachcontainadifferentshRNA.Thesecanbetransferredintocellstoseewhatphenotypesoccur.

E.RegulatoryRNAsand

X-Inactivation

InmammalsfemalesareXXandmalesareXY.Havingtwiceasmuc