Yous

afMa
nzoo
r3029

UNI
VERSI
TYOFOKARA

Mol
ecul
arBi
ology

Subj
ect
:Vacci
nol
ogy

Submi
tt
edt
o:Dr
.Tehmi
na

Submi
tt
edby:

YousafManzoor

Rol
lno:
3029

BS(
HONS)MOLECULARBI
OLOGY

er7th
Semest

①
 Yous
afMa
nzoo
r3029

Mi
croRNA(
miRNA)me
cha
nis
m
I
ntr
oduc
tion
Defi
nit
ion;“micr
oRNAi sasmallnon-
codingRNAmole
cul
etha
tfunc
ti
oni
nRNAs
il
enc
ing
andposttr
ans
cri
pti
ona
lre
gul
at
ionofgeneexpres
sion.
”

Hi
st
ory

Fi
rstmiRNAwa sdi
scove
redin1993byagrouple
dbyAmbr os.It
smodeofa cti
onpublic
ati
on
waspubli
shedbyRuvkun’st
eam.In2000and2ndsmallRNAwa schar
act
eri
ze.Thefi
rsthuman
di
seas
esassoci
atedwi
thdere
gulat
ionofmi
RNAwa sc
hroni
clymphocyt
icl
eukemia.

Modeofs
il
enc
ing
Sil
enci
ngmayoccurei
therthr
oughde gr
adat
ionorpre
ve nt
ingmRNAf r
ombeingt
ransl
ate
d.The
re
lat
ionofmiRNAa nditstar
getisbaseonnegat
iveregul
a t
ionoft
argetmRNA.He r
ecommon
sce
nari
oisus
ei-efe
ed-forwardloop.

Bi
oge
nes
isa
ndme
cha
nis
mofmi
RNA:
Theinvest
igat
ionoft hebiogenes
isandmecha
nism ofmiRNAss ti
llisinit
sinfa
ncy.Si RNA
appe
arstobeaf orm ofmiRNAdupl expr
edominan
tlyoccur
ri
ngi nplant
sandlowera nimals
.
Thebiogenes
isa ndme c
hanismofs i
RNAsareverysi
mil
artothoseofmi RNA.Howe ver,the
re
ares
omedi ff
erencesbet
wee nthe
setwopat
hways.Fi
veste
psareinvolve
di nmiRNAbi ogenesi
s
i
nve r
tebr
ates
.

 Fi
rst
,miRNAi
sge
ner
ate
da salongpr
imaryprecur
sormi
RNA(pr
i-
miRNA) ,mos
tli
kely
medi
ate
dbyRNApol
yme r
asestypeI
I(Pol-
II)
.Thepri-
miRNAi
stra
nsc
ribedfr
om the
ge
nome.

 Second,thelongpri-miRNAi sexc
ise
dbyDr osha-l
ikeRNa seII
Iendonucl
eas
esand/or
spl
iceosomalc omponentstoformthe*60–70ntpr e c
ur s
ormiRNA (pre
-miRNA).The
pre
-mi RNA e xhi
bit
sc onsi
derabl
eseconda
rys t
ruct
urei ncl
udi
ngregi
onsofimperf
ectly
pai
redds RNA,whi cha res e
quent
ial
lyclea
vedt ooneormor emi RNAs .Thi
ss t
e p
dependsont heor igi
noft hepri-
miRNA,whe t
herl ocat
edina nexonora nintr
on
res
pective
ly.

 Thir
d,t hepre-
miRNA i
se x
por
tedoutofthenucleusbyRan-GTPandareceptor,
Export
in-5.I
nthecyt
opl
asm,Di
cer-
li
keendonuc
lea
sescl
eavet
hepre
-miRNA t
of orm
matur
e18–25ntmi RNA.

Thema
tur
emi
RNAi
sinc
orpor
ate
dint
oar
ibonuc
lea
rpa
rti
cle(
RNP)t
ofor
mtheRNA-
induc
ed

②
 Yous
afMa
nzoo
r3029

genesi
lenci
ngcomple
x(RISC)whic
he xecut
esRNAi-
rel
ate
dgenes
il
enc
ing.Onl
yoneoft
he
twost
randsist
hemiRNA;t
heothe
rcounte
rpar
tisna
medmiRNA*.

ThematuremiRNAc anbl oc
kmRNAt ransla
tionbasedonpa r
ti
alcompl e
me nt ar
it
ybe t
weenthe
miRNAa ndtheta r
gete
dmRNA,pa r
ti
cul
a r
lyvi abas
epa i
ri
ngwi t
ht he30-untransl
atedreg
ionof
t
hemRNA.I fthereisape rf
ectcomple
me ntar
itybetweenthemi RNAa ndt het ar
getedmRNA,
mRNA de gr
ada t
ionoc curssimil
arl
yt ot ha
tme di
atedbys iRNA.Aut o-regulat
orynegati
ve
fe
edba
ckvi ami RNAsr egul
atessomege nes,incl
udingthoseinvolve
di nt heRNA s i
lenc
ing
mecha
nismitse
lf.

Theme
cha
nis
mofa
cti
onofmi
croRNAs
:
Theme cha nism ofa ct
ionofmi c
roRNAsi sc onsideredtobebyt womode s– tr
a nsl
ational
repress
iona ndt a
rgetde gradat
ion( Fi
gure-
5).Thef ormeri sc ommoni nma mma l
ians ystems
whi l
ethel atte
risfoundpr edomi nantl
yinplants
.Theba si
cdi f
fe r
enceinthetwome chanismsi s
thoughttobepr i
ma ri
lygove rnedbyt helevelsofc omplement ar
it
ybe tweenmi cr
oRNAsa nd
theirtargett ranscri
pts
.Pe rf
ectorne arpe r
fectc ompl ementari
tya si sc ommon i n pl ant
mi cr
oRNAs a ndi nas ma llc l
assofe ukaryoti
cmi croRNAsc ause
st ar
ge tcl
ea vagea nd
degradati
on131a nalogoust othea cti
onofs iRNAswhi c
hha vepe rfectcomplementarit
yt ot he
targetregions.Evi dences uggeststhatmicroRNA boundt ranscri
ptsares eques
tra
te dintoP
bodies132,133whe r
ethe ya r
ema inta
inedinas i
lence dst
ateeitherbya ssoc
iati
ngwithp roteins
thatpreventt r
ansl
ationorpos si
blybyr emovalofthec apstruc
ture133.

Tr
ans
lat
iona
linhi
bit
ion
:
Thef irstme chani st
i canal
ysesofmi RNA f unc t
ionwe r
ec a rr
iedoutus ingC.e l
egans:itwa s
foundt ha tthea bunda nceofmi RNA- regula
tedmRNAswa snots ubs t
a ntiall
yha nged,b utthe
abunda nceofpr ot einsencodedbyt hosemRNAswa sma r
ke dl
yr educ ed134, 135.Fur t
hermor e
,
t
her e
gula tedmRNAss eemedtobep rese
ntinpol ysome s134,135a nde nga gedwi t
hr i
bosome s
capableofs ubs eque ntel
ongati
oninvi t
ro135.The sestr
ikingobs erva t
ionss uggestedthatmiRNA
me diat
e ds uppr essionofpr otei
npr oducti
onoc c
urre
dbyame cha nismt hatope rat
e daft
ert he
i
nitiat
ionofpr oteins ynthe
sis
.Althoughs ubsequentstudiesha ves hownt hatmi RNAsc ane xer
t
t
he ireffectsbyava rie
tyofme chanisms,therea r
en ownume rouse xa mpl esinwhi chmiRNA-
me diat
e dr educ tionofpr ote
inproduc t
ioni snota c compa niedbyc orr espondingc hangesi n
mRNAa bunda nc e136-140.Inmos tofthe s
ec ases,t
het ranslat
iona ls t
atusofr egula
tedmRNAs
(i.
e.whe t
he rthe ya rea s
socia
tedwithpolysome s)hasnotbe ende termi ned.

Neve
rthe
les
s,t
hreerec
entre
port
sprovi
deevi
denc
eforr
epr
ess
ionofpr
ote
inpr
oduc
ti
ona
fte
r
pr
ote
insynt
hes
ishasbe
enini
ti
ate
d141-
143.

 Inthefi
rstst
udy,ami RNA-
regula
tedmRNA i nHeLacell
swasfounde
xcl
usi
vel
yin
pol
ysomesandassoc
iat
edwit
helongat
iona
llyc
ompet
entr
ibosome
s141.

③
 Yous
afMa
nzoo
r3029

 Int hes e
c onds tudy,ar epor
termRNAr esponsi
vetoa nendogenousmi RNAwa salso
pres
e ntinpol ys ome s142.Severa
ll i
ne sofe vi
denceshowedt ha
tt hes
epolysomeswe re
act
ive l
ytra nsl
ating;howe ver
,nas c
e ntpol
ype pti
descoul
dnotbede t
ect
edbyi mmuno-
pre
c ipi
tat
ion,s ugge s
tingt hatrepressi
onofpr ote
ina cc
umul a
tionr es
ult
edf r
omc o-
tr
anslati
ona lprote i
nde gra
da t
ion(
i.e.prot
einde gr
adat
ionconcomitantwit
htrans
lat
ion)
.

 I
nt hethir
dst
udy,arepor
terconstr
uct
scont
aini
nga3‘-
UTRwi thdesi
gnedtar
gets
itesf
or
as ynt
heti
cmiRNAt hathasextensi
vebutimper
fectc
omple
me nt
ari
tytothosesi
teswas
shownt obet
rans
lat
ional
lyre
p r
essedandas
soci
ate
dwithpol
ysomes143.

Va
cci
neswhi
chha
veus
edmi
RNAt
echnol
ogy
:
Vacc
ineshavebe
enhighl
ysucces
sfulatli
miti
ngvi
rali
nfe
cti
onsandhavele
dtot heel
imi
nat
ion
ofs
ma l
lpoxfr
omthehumanpopula
tion.Ther
earef
ourmaincl
ass
esofvir
alvac
cines:

 ki
ll
ed

 s
ubuni
t

 mRNA

 Li
ve-
att
enua
ted.

Live-
att
enua t
edvacc i
neshaves everaladva nta
ges,incl
udingpr ovidingmor erobust,long-l
asti
ng
immunityc omparedtoinactivatedvaccines.Vi r
usescanbea tt
enuate dthroughs everaldif
ferent
me c
hanisms,incl
udinga lt
eri
ngt hetempe ratureoftheoptima lpolyme rasefuncti
ona ndd el
et
ing
ormutatingviralimmunea ntagoni
sts.Me chani
smsofa ttenuationma ynotbei denticalacr
os s
vir
alspec i
es.Fore xample,theme chanism oft empera
tures ensiti
vi t
ywi llvaryf rom virust o
vir
us.Addi t
ional
ly,safetyisama j
o rc oncernwhe nde velopinga nyl i
ve-att
enua t
edv a
ccine,
wherereversi
ontowi ldtyper e
plica
tionc ouldbede va
stat
ing.

Ther
oleofmi
RNAsi
nthei
nna
tea
nti
-vi
ralr
espons
e

Thehos tinnateimmuner e spons eisthefir

stli
neofde fe
nsea gai
ns tallpat
hoge ns.Al argevariety
ofc ells
,s ucha sepithel
ialc el
ls,dendrit
icc ell
s,granuloc ytes,monoc ytes,ma cropha ges,a nd
naturalkill
ercell
s,playani mpor ta
ntroleinthei mmuner e sponse .Somemi RNAsa reproduc ed
duringvi ralinfe
c t
ion,modul atingthef unctionofa l
lt hepr e viousl
yme nt i
one dc e
lls
.Vi r a
l
gene t
icma t
eri
altri
gge r
sa ctiva t
iono fthehos tinnateimmuner esponses
,a ndt hisma t
eri
a lis
recognizedbys everalpatt
e rn-recognit
ionreceptors(PRRs ),s ucha sNod- l
iker ece ptors(NLRs )
,
RIG- li
ker ece
ptors(RLRs ),a nd,mos timportantl
y,Tol l
-l
iker eceptors.Thei nteractionofg ene ti
c
ma teri
alwi t
he achoft heser eceptorsdependsonwhe t
he rt hema ter
ialisDNA orRNA a nd
whe thertheRNAi ss i
ngles trandedordoubl es t
randed.Itiswe l
lknownt hatr espiratoryviruses
ma inlyinter
actwithRLRs( suc hasRI G-IandMDA5a tt hec ytoplasmicleve l)andwi thTLRs
(s
uc ha sTLR3,TLR7,TLR8,a ndTLR9a tthee ndosomi cl evela swe l
la sTLR2a ndTLR4a t

④
 Yous
afMa
nzoo
r3029

t
hesur
faceofthec e
llme mbr
ane)
,wh ichculmina
tei
nt wosignal
ingpat
hwa ys
.Thef i
rstt
ri
ggers
a
cti
vat
ion oft he NF- κB tr
ans
cription f
actor
,whi chiniti
atesthe t
ranscr
ipt
ion ofpr o-
i
nfl
ammatorycytokines
,whil
ethes e
c ondisli
nkedt
oa ct
ivati
onofinte
rfe
ronregul
a t
oryfac
tors
(
IRFs)
,whichpromot et
ypeIandIIIinter
fer
ongeneexpres
sion

mi
RNAva
cci
nes
:
mi RNAsha vebe enus eda saplatf
ormt ode vel
opl i
ve-att
enua tedvaccinesforDNAa ndboth
pos i
tiveandne gati
ves enseRNA vi ruse
sbye xploi
tinghos te ndogenousmi RNAs .Thebroad
arrayofc ell
-a nds pecies-
speci
fi
cmi RNAs ,a swe l
last hea bil
it
yt ot ar
getusingmul t
ipl
e
mi RNAs ,allow forc ustomiza
bleatt
enuationfordiffere
ntvi ruses.A ma joradvanta
geoft hi
s
st
ra te
gyi sthatt
hispr ovidesaknownme chanismofa tt
enuation,whi chc a
nbea pplie
da c
rossa
dive r
serangeofvi ruses.Inthi
sreview,wewi l
ldiscussthea pproachesforgenerat
ingmiRNA-
ta
r getedv i
rusesa nds e ver
alstra
tegi
esf ori mprovingt hes a f
etya nde ff
icac
yofmi RNA-
at
te nuate
dva cci
nes.

Me
cha
nis
msofMi
croRNATa
rge
tingofVi
rus
es

Endogenousmi
RNAt arget
ingprimar
il
yoccur
satthethr
eeprimeuntr
ans
lat
edr egi
on(3’UTR)
ofhostmRNAt r
ansc
ripts
.Howe ver
,manyvir
alRNAsc ont
ainshor
t3’UTRs ,pote
nti
all
yasan
evol
uti
onaryme
chani
smt oe va
demiRNAr e
gulat
ion.

The rea retwowa yst ha tvirusesc anbee ngi ne eredt obes ensitivet omi RNAs :throu ghthe
gene rat
ionofs ile
ntmut at
ionsint heope nr eadingf r
a met obec ompl e
me nta rytothec ognat
e
mi RNA orbyi nsertingc ompletet argetsit
e sint ot hee ndoge nousore ngi nee redUTRs .Some
locationsi nt hevira lge nomema ynotbea me nablet omi RNA ma chinerya ccessduet othe
seconda ryRNAs truc tur eorbindi ngofvi ralpr oteins,andt herefor ecannotbeus edtoa t
tenuat
e
virusr epli
ca t
ion.We stNi levirusha sbe e
ne ngine eredt oe ncodemi RNAt ar getsit
esb e t
ween
RNAs econda rystr
uc tur esinthe3’UTRt oa l
lowmi RNAa cc essa ndtopr eve ntdi s
ruptionofthe
impor tantRNA s t
r uc tures.The ser es
ultsde mons trat
et hatc ar
emus tbet akeni nc hoosi
ng
genomi csit
e sformi RNAt ar
geting.I naddit
iont othel ocati
onwi thinage ne ,e ngineer
edmi RNA
-me diatedre press
ionc anbea f
fe ctedbyt hec hoi c
eofvi ralge nebe ings il
enc e
d.Ta rgetingan
essentialge nec anc ompl ete
lydi sruptvirusr epl ic
a t
ion,whi l
et het argetingofanon- essent
ial
genec anpre ventpa thol ogybuta llowf orthee xpre s
sionofs omevi r
alpr oduc ts
,whi chma ybe
nec essarytopr oducear obustada ptiveimmuner espons e.Re versi
ont ovi rulenc eisac once rnf
or
alllive-att
enua t
edva cc ines,i
ncludi ngmi RNA- t
a rgetedva cci
ne s

Ne
wthe
rapi
esf
ocusonmi
RNAsa
ndt
hei
rut
il
it
yinva
cci
nes

Curr
ent
ly,nodr uge xi
ststhati
ncrea
sestheleve
lsof ,orinhibi
ts,anymi RNAsi nvir
a lARI s
;
howeve
r,ther
eha sbe e
ns omeprogre
ssonotherdi
s e
a s
es.Thef i
rsti
nhibi
torydrugforas peci
fic
miRNA( miR-122)wa screat
edin2010and,asofthiswrit
ing,isi
npha seIItr
ial
sforhepa t
it
isC
tr
eat
ment.Thef i
rstsynthet
icmi RNA,mi R-34( MRX34) ,wa sde vel
opedi n2013f ort he

⑤
 Yous
afMa
nzoo
r3029

t
rea
tme
ntofa
dva
nce
dhe
pat
oce
llul
arc
arc
inoma
.

Inmor er ecentr esearch,s yntheti

cmi RNAsha vebe enge ne rate
dtha ta rec arri
edbyl ipos ome s
andt rans f
ectedi ntot hemononuc l
e a
rc el
lsofpe ri
phe r
a lblood.The sepr otoc olsinc
reasec e r
tai
n
pro-inflamma t
oryc ytoki nes,sucha sTNF- α,favoringt hei nnateimmuner esponse
.Themos t
recenta ppli
cati
onoft he s
emi RNAsha sbe ent hec re a
tionofne w va cci
ne swi t
ha tt
enua te
d
virusest hata rel oade dwi t
ha ne xpres
sionc ass
ettee ncodi ngas ynt heti
cmi RNA t ha ttargets
st
ruc turalpr ote
insoft hevi rus.ThePR8- a
mi R-93NPvi ruswa sge ne r
ate dbyi nse r
ti
nga n
expr ess
ionc assettef ormi R-93be tweenvi ralge nese nc odingnon- s
tructuralproteinsi na n
att
e nuatedI V,a ndt hismi RNAs pecif
icall
ytargetsthenuc leoprot
einsoft heI V.Thi sv accine,
admi nisteredintrana sally,conf e
rredimmuni t
ya gainsts e
ve ralhete
rologousvi ralstr
ains.Pl ants
alsopr oduc emi RNAst hatregulatevirusrepli
c a
tion.Ane xampleisMI R2911i nhone ysuckle,
whi chi nhibitsthee xpre ss
ionoft hePB2a ndNS1pr otei
nsoft hei nfluenzaA vi rusesH1N1,
H5N1a ndH7N9.

Conc
lus
ion:

miRNAspl ayacruci
alr
oleintheregul
ati
onof( infa
vorofora gains
t)thei nna
tei
mmune
r
esponseinvir
alARIs
.Overa
ll,mi
RNAt arget
ingisapromi
si
ngpla t
formforde vel
opi
ngsafe
,
ef
fect
ivevacci
nesandprovi
desinc
rease
dpl ast
ic
ityovert
radi
ti
onall i
ve-
a t
tenuat
edvac
cine
st
rat
egie
s.