Antisense RNA technology & its role in crop improvement ppt surendra singh

Antisense RNA Technology & Its Role in Crop Improvement

2. Speaker: SurendraSingh
Id No. 40925
Antisense RNA Technology & Its
Role in Crop Improvement

3. Introduction
AntisenseRNAtechnologyisamethodthatusedfortheinhibitionof
geneexpression.
Theantisensenucleicacidsequencecanbeeithersynthetic
oligonucleotidesof30nucleotides(nt)orlongerantisensesequence.
Thistechnologymayusedfortherapeuticpurpose,functionalgenomics
andtargetvalidation.
AntisenseRNAtechnologycanbeusedtopreventviraldisease.
AntisenseRNAtechnologyusedforimprovementofqualitycharacters
inplants.

4. Antisense RNA approach
AntisenseRNAisasinglestrandedRNAthatis
complementarytomessengerRNA(mRNA)transcribed
withinacell.
Therearemanycasesinbothprokaryotesandeukaryotes
whereasinglestrandedRNAbasepairwitha
complementaryregionofanmRNAanditsprevent
expressionofthetargetmRNA.
Sensestrandandantisensestrand:
referstotheoriginalsequenceofDNAorRNA
moleculewhile,antisensereferstothecomplementary
sequenceofDNAorRNAmolecules.

5. Types of Antisense RNA Approach
NonCatalyticantisenseRNA
NoncatalyticantisenseRNAstrandsblocksRNA
processingi.e.–modifiedtheRNAstrandsor
transcription
CatalyticantisenseRNA
CatalyticantisenseRNAalsocalledribozymeswhichwill
cleavethemRNAmoleculesatspecificsequence

6. Construction of antisense RNA
•AntisensegenesorRNAareconstructed
byreversingtheorientationofgenewith
regardtoitspromoter.

7. Mechanism of non catalytic antisense gene
•Threetypesmechanisminantisensegenepreventgene
expression.
•mRNAnotavailablefortranslationmachinery.
•DegradationbydsRNAspecificenzymeRnaseHwhichbreaks
phosphodiesterbondanddegradestargetmRNA.
•ItrepressthesplicingprocessinpremRNA.

8. Catalytic Antisense RNA or Ribozymes
RibozymesareRNAenzymesthathaveendoribonuclease
activity.
Ribozymesusedforgeneknockdownapplicationhaveacatalytic
domainthatisflankedbysequencecomplementarytothetarget
RNA.
OncetargetRNAdestroyedribozymesdissociateand
subsequentlycanrepeatcleavageonadditionalsubstrate.
FirstisolatedfromviroidRNAs.
RibozymeactivityenhancedthroughhighMg
++
andhighamount
oftargetRNA.

9. RNA interfernce(RNAi)
•RNAiisaadvancedtechniqueofantisenseRNAtechnology.
•RNAiisanaturalpowerfulmechanismthatisthoughtto
havearisenforprotectionfromvirusesandtransposon.
•RNAifirstdiscoveredinnematodeC.elegansbyAndrewz.
fireandCraigmelloandgetnobelprizein2006.
•RNAistopthegeneexpressionthroughcomplementory
smalldoublestrandedRNAmolecules.
MostlytwotypesofRNAimoleculesstudies.
•SmallinterfernceRNA(siRNA).
•microRNA(miRNA)

10. COMPONENTS OF GENE SILENCING

11. Key enzymes in gene silencing:
Haruhiko Siomi& MikikoC. SiomiNature/vol457/22 2009

12. DICER A TYPE III RNA-SPECIFICRIBONUCLEASE
EnzymeinvolvedintheinitiationofRNAi.Itisabletodigest
dsRNAintouniformlysizedsmallRNAs(siRNA)
DicerfamilyproteinsareATP-dependentnucleases.
RnaseIIIenzymeactsasadimer
DicerhomologsexistinmanyorganismsincludingC.elegans,
Drosphila,yeastandhumans
Lossofdicerleadstolosssilencingprocessinginvitro
FourkindofdicerinArabidopsisthaliana
DCL1-generationofmiRNA
DCL2-ProduceviralderivedsiRNA
DCL3-GenerateDNArepeatassociatedsiRNA
DCL4-SynthesisoftasiRNa

13. AGO Protein Family
RNAbindingproteins,throughconservedPAZdomain
AssembleintoRISC
DirectcleavageoftargetmRNA
10AGOmembers
FunctionaldiversificationofRNAsilencingislinkedtothe
variationbetweenAGOfamilymembers
e.g.,AGO1-associatedwithmiRNApathway,AGO4with
endogenoussiRNAsilencing

14. The RNA-induced silencing complex
(RISC)
MulticomponentRNAinuclease(500kDa):
1.MemberofArgonautefamily
2.RNAbindingproteins
3.RNAhelicase
4.Ribosomalprotein
Sequence-specificnucleasethatusessmallRNAas
guidestotargetspecificmessages basedupon
sequencerecognition.
CleavagesiteofcognateRNA(mRNA)isplacednearthe
middleoftheregionboundbythesiRNAstrand.

15. Small RNA Molecules

16. The small interference RNA
(siRNA)
•siRNAmadeartificiallyorproducedinvivofrom
doublestrandedRNAprecursors.
•21-25nucleotideslong.
EachstrandofsiRNAhas:
a.5’-phosphatetermini
b.3’-hydroxyltermini
c.2/3-nucleotide3’overhangs

18. miRNA
21-24ntinlength
ThemiRNAareendogenoussmallRNAguidesthatrepress
theexpressionoftargetgenes.
DifferfromsiRNAinbiogenesisnotinfunctions,although
mechanisms canbedifferent.mRNA cleavewhen
complementaritiesisextensive,represstranslationwhen
not.
Morethan100miRNAinArabidopsisessentialforplant
development.

19. miRNAbiogenesis

20. TranslationofcellmRNAissuppressedbyits
complementarypairingwithmiRNA.
ItisknownthatmiRNAsareproducedfromcell
untranslatedtranscriptsof120–150ntandeven
producedbyDICERfromamiRNAprecursor.
Thisinteractioninducesdegradationofthetarget
mRNAordirectlyblockstranslation.
Endogenous mRNA Silencing Caused by miRNA

21. Comparison of siRNAand
miRNA

22. Other types of siRNA
tasiRNA
Chromatin associated siRNA
natsiRNA
scnRNA
rasiRNA
lsiRNA
snoRNA
piRNA

23. tasiRNA
21-22nt long
Generation initiated by miRNA and or require DCL4 & RDR6
Role in plant development and hormone signaling
Down regulate auxin response factors

24. Chromatin assisted siRNA
24-25ntlong
MostabundantclassofsiRNA
Generated by DCL3/RDR2/RNA PolIV/AGO4 to
transcriptionalsilencing
RoleinDNAmethylation&transcriptionalgenesilencing

25. natsiRNA
EndogenoussiRNAderivedfromtheoverlapping
regionofapairofnaturalantisensetranscripts
(NATs).
20-21ntlong.
RegulatesaltstressresponseinArabidopsis.
Majorsourceofendogenous siRNAforgene
regulationinresponsetodifferentenvironmental
conditions.

26. scnRNA
Protozoan
First Identified in Tetrahymenathermophyla
27-30nt long
Role in developmentally programmed DNA
elimination (Lee and collins, 2006)

27. piRNA
Mammaliangermlines&stemcells
26-31ntlong
piRNAattacksatranscribedtransposon&the
cleavedproductcontributetothepiRNA
production(Aravinetal.,2006)

28. rasiRNA
Drosophila
24-29 ntlong
Share common features with piRNA

29. lsiRNA
30-40ntlong(longsiRNA)
SharecommonfeatureswithendogenoussiRNA
Inducedeitheronpathogeninfectionorspecificgrowth
conditions
DownregulationofgeneexpressionintotheformofmRNA
decappingor5’-3’-degradation

30. Daniela Castanotto & John J. Rossi. Nature/vol/457/22 ,2009

31. Application in Crop Improvement

32. Slow Fruit Softening in Tomato
•InTomatoenzymepolygalacturonase(PG)degradespectin
whichisthemajorcomponentoffruitcellwall.
•Itleadstothesofteningoffruitsanddeterioratethefruit
quality.
•IntransgenictomatoexpressionofPGantisenseRNA
dramaticallyinhibitPGmRNAaccumulationandenzymeactivity
Theologis et al., 1992

33. Delayed Fruit Ripening in Tomato and Flower
Senescence in Carnation
•Fruitripeningandflowersenescenceis
promotedbyethylenephytohormone.
•Ethylenebiosynthesisinvolvestwoenzymes
viz-ACCsynthase(encodedbyaccgene)and
ACCoxidase(acogene)
•Methionine AdoMet ACC Ethylene
•Geneaccwassuppressedbyusingantisense
ofitscDNA.
•Thatantisensereducedmorethan90%of
ethyleneproduction.
Hamilton et al.,1992

35. Male Sterility and White Flower Production in Petunia
•Flavonoidsareessentialfornormalpollendevelopmentand
function.
•Chalconesynthaseisakeyenzymeforflavonoidessynthesis.
•InPetuniaantisenseofenzymeCHSisinsert.
•TransformedplantsofPetuniahavenegligibleCHSactivityand
producewhiteflowerandnonfunctionalpollen.
•Formaintenanceofmalesterilelinewegiveflavonolchemical
duringpollendevelopment.
Meer et al., 1992

36. Changed Fatty Acid Composition of BrassicaOil
Enzymestearoyl-ACPdesturasecatalyzetheconversionof
stearoylACPtooleoyl-ACP.
TransgenicB.rapaandB.napusplantscontainingthe
antisenseRNAofB.compestrisstearoyl-ACPdesaturase.
Transgenicplantshighlyreducedlevelofoleicacidand
increasethelevelofstearicacidfromlessthan2%toupto
40%.
Thesemodificationareaimedtogeneratingsaturatedfatty
acidnowobtainedfromcocoa.
knutzon et al, 1992

37. Protection of Plants From Viral Infection
TransgenicBarleyresistanttoBYDV(Barleyyellow
dwarfvirus)(Wangetal.,2000).
TransgenicBananaresistanttobananabractmosaic
virus(Williamsetal.,2004).
InbananadesigningRNAivectoraimedatsilencingthe
coatproteinregionofthevirusthatisresistantto
BBrMV.

38. Production of Low Glutenin Rice
ThericemutantlineLGC-1(LowGlutelinContent-1)
wasthefirstcommerciallyusefulcultivarproducedby
RNAi(Kusabaetal,2003).
Itisalow-proteinriceandisusefulforkidneypatient
unabletodigestgluteninprotein.

39. Reduced Lignin Content in Jute
ApossibleapplicationofRNAiinvolvethedown
regulationofakeyenzymeinbiosyntheticpathwayof
lignin.
Theenzyme4-coamarateligase(4-cl)isatypeofkey
enzymeinearlystageofligninsynthesis
TransgenicjutevarietyexpressingRNAiconstructto
downregulatethequantityof4-clmRNA.
Williams et al., 2004

40. Reduced Gossypol Content in Cotton
Cottoncantainsatoxicgossypolterpenoid.Itprotects
cottonplantsfromattackofinsectsandotherpathogens
butitisharmfulforbothanimalandhuman.
TransgeniccottonplantexpressingaRNAiconstructofthe
d-cadinenesynthasegenetofusedwiththeseedspecific
promotercausedseedspecificreductionofgossypol
metabolites.
kumar et al., 2006

41. Increasing Grain AmyloseContent in Wheat
Foodrichininefficientlydigestedcarbohydratessuchas
fiberareconsideredtobehealthpromoting.
Themajorsourceofplantderivedcarbohydrateis
starchwhich,iscomposedofamylopectinandamylose
polysaccharides.
Forincreasingtheamylosecontentinwheatgrainsa
RNAiconstructdesigntosilencethegeneencoding
starchbranchingisozymesofamylopectinsynthesis.
Thisresultincreaseamylosecontentinwheatgrainby
70%oftotalstarch.
Tang et al., 2006

42. Reduced Neurotoxin in Khesari
Khesari(lathyrussativus)isalegumecrop.
KhesaricontainaneurotoxinBOAAwhichcauses
paralysisinlowerlimbscalledlathyrism.
RNAitechnologycanbeusedtosilencethegene
responsibleforBOAA.
BOAAgenesmaybelinkedtothisuniquecropwith
droughtandfloodtolerance.
So,bringingdownthelevelsofBOAAtosafe
concentrationratherthentotallysilencedthegene.
Williams et al.,2004

43. Reduced Caffeine Content in Coffee
Caffeineisastimulantofthecentralnervoussystem.
Itsadverseeffectincludeinsomaniaandrestlessness.
10%ofcoffeeontheworldsharedbydecaffeinate
coffee.whichhave2mgto4mgcaffeinepercupwhile
standardcupoffiltercoffeegenerallycontains60to150
mgcaffeine.
RNAitechnologyhasenabledthecreationofvarietiesof
coffeethatproducedlowcaffeinecontent.
Van Uyen, 2006

44. Production of Blue Rose
FlowercolourinplantsbyAnthocyanin
ViolettobluecolourisduetoDelphinidinandred
colourduetocyanidinanthocyanin
RNAitechniqueusedforcyanidingeneknockdown
Anewdelphinidingeneintroducedintorosewhich
convertedredcolourintobluecolour.

45. How to make a blue rose

46. Improve Fruit Quality by RNAi
Broothaertsetal.,(2004)reportedtheproductionof
transgenicappletrees.Theyareabletoselfpollinate
anddevelopfruits.
ThisbreakthroughwasachievedbysilencingtheS-
generesponsibleforselfincompatibility.
Transgenicapplefruitssilencethekeyenzyme
autocatalyticethyleneproductionwasdisplayedan
increasedshelflife(Dandekaretal.,2004).
Guoetal.,(2005)studiedthatthetransgeniccitrus
downregulatetheputativepectinmethylesterasegene.

48. Advantages
Highly gene specific
High gene silencing efficiency
Screening targeted plants takes less time
Highly inducible
Disadvantages
It does not knockdown a gene for 100%
siRNA tends to activate unwanted pathways
--Expensive
--Ethical problems

49. Conclusion
BasedonpresentdiscussionantisenseRNAtechnologyisa
efficientknockdowntechnologyinplants.
Itisthoughttobeusefulforgeneticimprovementevenin
plantswithlowtransformationefficiency.
BasicconceptoftheapplicationoftransgenebasedRNAito
thegeneticimprovementofcropplanthasbeenestablished.
Furtherstudiesareneededforitswiderapplication