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文檔簡(jiǎn)介
Non-coding
RNAs1.History
and
discoveryiescher.Nucleic
acids
were discovered
in
1868
byFriedriTogetherwith
DNA,
RNA
comprisesthe
nucleic
acids.Friedriiescher
(1844–1895)Messenger
RNA(mRNA)Robert
W.HolleyHar
Gobind
KhoranaMarshall
W.NirenbergDiscovery
of non-coding
RNA-
alanine
tRNAThe
role
of
RNA
in
proteinsynthesis
was ed
already
in1939.In
1965,
Robert
W.
Holley
et
al,
found
the
non-coding
RNA
characterised
asanine
tRNA
found
in
baker's
yeast.1History
anddiscoverySuggested
secondary
structure
of
the
alanine
transfer
RNA(NobelLecture,
December,
12,
1968)X-ray
t-RNA
Ala原核生物的rRNA分三類(lèi):5SrRNA、16SrRNA和23SrRNA。真核生物的rRNA分四類(lèi):5SrRNA、5.8SrRNA、18SrRNA和28SrRNA。Three-dimensional
representationof
the
50S
ribosomal
subunit.rRNA
isin
ochre,
protein
in
blue.TheNobel
Prize
in
Chemistry1989
was
awarded
jointly
to
SidneyAltman
and
Thomas
R.
Cech"fortheir
discovery
of
catalyticproperties
of
RNA".Thomas
R.CechSidney
Altman26SrRNAElectron
microscopy
imagesof
the
yeastspliceosomesnRNAs,combined
with
protein,
form
splicesome表觀遺傳學(xué)課上的兩類(lèi)ncRNA1. Small
ncRNAs(small
silencing
RNA)2. Long
ncRNAs2.Small
ncRNAs(Small
silencing
RNAs)Although
many
classes
of
small
ncRNAs
have
emerged,
various
aspects
of
their
origins,structures,
associated
effector
proteins,
and
biological
roles
have
led
to
t
eralrecognition
of
three
main
categories:small
interfering
RNAs
(siRNAs),
microRNAs(miRNAs),
and
piwi-interacting
RNAs(piRNAs).What
ismicroRNA?The miRNA,
lin-4
from
Caenorhabditis
elegans,
was
discovered
by
Ambros
andcoworkers
in
1993as
an
endogenous
regulator
of
genes
that
control
developmentaltiming.
Mature
microRNA
is
approxima y
22
nt
in
length,
which
control
geneexpression
mostly
at
the
post-transcriptional
level,
in
metazoan
animals
andplants.MicroRNA,
miRNALin
He
and
Gregory
J.Hannon.
Nature
Reviews
Genetics
5,
631
(2004).miRNA
gene
familiesmiRNAs
with
identical
sequences
at
nucleotides
2–8
ofthe
maturemiRNA
belong
to
the
same
‘miRNA
family’
.Some
miRNAs
share
a
common
evolutionary
origin
butdiverge
in
the
miRNA
seed.hsa-let-7ahsa-let-7bhsa-let-7chsa-let-7dhsa-let-7ehsa-let-7f6
-
ugagguaguagguuguauaguu-
276
-
ugagguaguagguugugugguu-
2711-
ugagguaguagguuguaugguu-
328
-
agagguaguagguugcauaguu-
298
-
ugagguaggagguuguauaguu-
297-
ugagguaguagauuguauaguu-28hsa-miR-141hsa-miR-200c59
-uaacacugucugguaaagaugg
-8044
-uaauacugccggguaaugaugga-
66miRNA
gene
nomenclaturemiRNAs
found
in
early
genetic
studies
werenamed
after
their
phenotypes
(for
example,
lin-4,let-7)miRNAs
found
from
cloning
or
sequencingreceived
numerical
names
(for
example,
the
lin-4homologues
in
other
species
are
called
mir-125)Genes
encoding
miRNA
sisters
are
indicated
withlettered
suffixes
(for
example,mir-125a
andmir-125b)Each
locus
produces
two
mature
miRNAs:
onefrom
the
5?
strand
and
onefrom
the
3?
strand
ofthe
precursor
(for
example,
miR-125a-5p
andmiR-125a-3p).one
arm
(called
the
‘guide’
strand)
is
usuallymu
ore
prevalent
(96–99%
of
the
sum
onaverage)
and
more
biologically
active
than
theother
arm
(the
‘passenger’
strand,
which
is
knownas
miRNA*).Julia
Winter,
et
al.
NatureCell
Biology,
11(3):228–234,2009The
canonical
pathway
of
microRNA
processing12345MicroRNA,
miRNAEarly
steps:
microRNA
processing
in
the
nucleusTranscription
ofthe
pri-miRNA.MiRNA
genes
are
transcribed
by
eitherRNA
polymerase
II
or
RNA
polymerase
III
into
primary
miRNA
transcripts
(pri-miRNA)MicroRNA,
miRNADGCR8
contains
two
double-stranded
RNA-binding s
for iRNA
processing.A age
human
pri-miRNA
contains
a
hairpin
stem
of
33
base-pairs,a
terminal
loop
and
twosingle-stranded
flanking
regions
upstream
and
downstream
of
the
hairpin.The
double-stranded
stem
and
the
unpaired
flanking
regions
are
critical
for
DGCR8
binding
andDrosha
cleavage.MicroRNA,
miRNAMirtrons:
splicing
replaces
Drosha
cleavageIntron-derived
microRNA
is
a
new
class
ofmiRNA
derived
from
the
processing
ofgeneintrons.Intron-derived
miRNAs
are
released
fromtheir
host
transcripts
after
splicing.
If
theintron
resulting
from
the
action
of
thesplicingmachinery
and
the
lariat
debranchingenzyme
has
the
appropriate
size
to
form
ahairpin
resembling
a
pre-miRNAMicroRNA,
miRNAPri-miRNA
cleavage
by
the
Drosha–DGCR8microprocessor
complexThe
pri-miRNA
is
next
endonucleolytically
cleaved
by
the
nuclear
microprocessor
complexformed
by
the
RNase
III
enzymeDrosha
(RNASEN)
and
the
DGCR8
(DiGeorgecritical
region
8)protein(also
known
as
Pasha
(Partner
of
Drosha)
in
D.
melanogaster
and
C.
elegans).The
microprocessor
complex
Drosha–DGCR8cleaves
the
pri-miRNA,
releasing
the
pre-miRNA.RNA甲基化DGCR8
directly
and
stably
interacts
with
thepri-miRNA
and
functions
as
a
molecular
ruler
todetermine
the
precise
cleavage
site.MicroRNA,
miRNADrosha
cleaves
11
base
pairs
away
from
thesingle-stranded
RNA/double-stranded
RNAjunction
at
thebase
of
the
hairpin
stem.MicroRNA,
miRNASelf-regulation
of
the
microprocessorcomplexThe
two
components
of
the
microprocessorcomplex
regulate
each
other.Exportin-5–Ran-GTP
mediate
the
export
of
thepre-miRNAMicroRNA,
miRNAExportin-5
recognizes
the
pre-miRNA
independently
of
its
sequence
or
the
loopstructure.A
defined
length
of
the
double-stranded
stem
and
the
3′
overhangsare
important
for
successful
binding
to
Exportin-5,
ensuring
the
export
of
only
correctlyprocessed
pre-miRNAs.Lee
SJ,
et
al.
Curr
Opin
Struct
Biol.
2011
Feb;21(1):101-8.MicroRNA,
miRNAComing
of
age:
microRNA
maturation
in
the
cytoplasmThe
RISC
loading
complex
(RLC):
Dicer,
TRBP
and
PACT
join
Ago2RLC
is
a
multi-proteincomplex
composed
of
the
RNase
Dicer,
the
double-stranded
RNA-binding proteins
TRBP
(Tar
RNA
binding
protein)
and
PACT(proteinactivator
of
PKR),
and
the
core
component
Argonaute-2
(Ago2),
which
also
mediates
RISCeffects
on
mRNA
s.TRBP
and
PACT
are
not
essentialfor
Dicer-mediated
cleavage
of
the
pre-miRNA(next
step)
but
they
facilitate
it,
and
TRBP
stabilizes
Dicer.Small
interfering
RNAss,Processing
occurs
most
readily
at
dsRNA
ends,
whichassociate
with
the
PAZ
(PIWI–AGO–ZWILLE)
present
in
most
Dicer
enzymes.
The
substrate
isthenpositionedwithin
the
active
sites
of
the
RNase
III
which
cleave
the
20
nt
siRNA
duplex
from
its
precursor.Dicer:
A
Portal
into
RNA
SilencingDicer
proteins
cleave
dsRNA
precursors
into
characteristiclengths
through
theaction
of
two
RNase
III
s.Cleavage
of
the
hairpin
intoa
duplex
by
DicerMicroRNA,
miRNAThe
RNase
III
Dicer
cleaves
off
the
loop
of
the
pre-miRNA
or
the
nickedac-pre-miRNA
andgenerates
a
roughly
22-nucleotide
miRNA
duplex.Argonaute:
At
the
Core
of
RNA
SilencingArgonaute
proteins
are
RNAsilencing
effectors
that
are
guidedto
their sby
short
single-stranded
nucleic
acids.
The
5’
endof
the
guide
strand
associateswith
a
binding
in
the
Mid,
and
the
3’
end
binds
thePAZ .
The
cleavagesite
isjuxtaposedwith
active-site
residues
in
thePIWI ,
though
in
this
casecleavage
is
suppressed
bymismatches
between
the
guideandthe
.MicroRNA,
miRNAGuide
strand
selection,
asymmetry
and
small
RNA
sortingIn
principle,
the
miRNA
duplex
could
give
rise
to
two
different
maturemiRNAs.In
a
similar
manner
to
siRNA
duplexes,
only
one
strand
is
usually
incorporated
into
RISCand
guides
the
complexto mRNAs;
the
other
strand
is
degraded.This
functional
asymmetrydepends
on
thethermodynamic
stabilityof
thebase
pairsat
thetwoends
of
the
duplex:the
miRNA
strand
with
the
less
stable
base
pairatits5′
end
inthe
duplex
isloaded
intoRISC.Cleavage
of
the
hairpin
intoa
duplex
by
DicerMicroRNA,
miRNAThe
RNase
III
Dicer
cleaves
off
the
loop
of
the
pre-miRNA
or
the
nickedac-pre-miRNA
andgenerates
a
roughly
22-nucleotide
miRNA
duplex.Ago2-mediated
pre-miRNA
cleavageFor
miRNAs
that
display
a
high
degree
of
complementarity
along
the
hairpin
stem,
anadditional
endonucleolytic
cleavage
step
occurs
before
Dicer-mediated
cleavage:
theslicer
activity
of
Ago2cleaves
the
3′
arm
of
the
hairpin
in
the
middle
to
generate
anicked
hairpin,
producing
the
Ago2-cleaved
precursor
miRNA
(ac-pre-miRNA)MicroRNA,
miRNAMicroRNA,
miRNAConclusions
and
outlook:
cellular
effects
of
microRNA-specific
processing
andpost-transcriptional
regulationMicroRNA,
miRNAThe
RISC
can
inhibit
the
expression
of
the mRNA
through
two
main
mechanisms
thathave
several
variations:
removal
of
the
polyA
tail
(deadenylation)
by
fostering
the
activity
ofdeadenylases
(such
as
CCR4–NOT),
followed
by
mRNA
degradation;
and
blockadeoftranslation
at
the
initiation
step
or
at
the
elongation
step;
for
example,
by
inhibitingeukaryotic
initiation
factor
4E
(EIF4E)
or
causing
ribosome
stalling
RISC-bound
mRNA
can
belocalized
to
sub-cytoplasmatic
compartments,
known
as
P-bodies,
where
they
are
reversiblystored
or
degraded.MicroRNA,
miRNAMost
plant
microRNAs
(miRNAs)
anda
few
animal
miRNAs
direct
endonucleolytic
cleavage(slicing)
of
their
mRNA s.The
5?-to-3?
exoribonuclease
XRN4
in
plants
and
XRN1
inanimals,
together
with
the
major
cellular
3?-to-5?
exonucleolytic
complex,the
exosome,subsequently
degrade
the
sliced
mRNA
fragments.In
animals,
miRNAs
were
originallyproposed
to
repress
translation
of
an
open
readingframe(ORF).
Biochemical
studies
have
suggested
that
miRNAs
have
a
role
in
blocking
translationalinitiation,in
poly(A)
tail
shortening
or
in
therecruitment
of
proteincofactors
that
caninterfere
with
translation.MicroRNA,
miRNAMicroRNA,
miRNAIn
many
cells
and
tissues,miRNA-directed
translational
repression
is
indistinguishablefrom
mRNA
destructionvia
decap and
5?-to-3?
decay.
This
hasled
to
the
suggestionthat
miRNAs
directly mRNAs
for
decay.Another
possibility
is
that
the
inhibition
oftranslation
by
miRNAs
triggers
subsequent
mRNA
decay,
and
the
temporal
delay
betweenthese
two
effects
can
vary
depending
on
the
surveillancemechanismsin
place
inparticular
cellular
contexts.miRNA的表達(dá)檢測(cè)miRNAmiRNAReal
timeQ-PCR檢測(cè)miRNA
發(fā)夾前體序列已升至24521
條,新增3
千余條。成熟miRNA
序列升至30424
條,新增5
千余條。人成小鼠成miRNA
新增至2578
條;miRNA
新增至1908
條;大鼠miRNA
新增至728
條。miRNA
mimic是運(yùn)用化學(xué)方法
的雙鏈RNA,能模擬細(xì)胞中內(nèi)源性成熟miRNA的高水平表達(dá),以增強(qiáng)內(nèi)源性miRNA的調(diào)控作用,進(jìn)行功能獲得性(gain-of-function)研究。只需直接轉(zhuǎn)染進(jìn)入細(xì)胞,即可檢測(cè)功能變化,快速,方便。LNA(Locked
Nucleic
Acid),通過(guò)化學(xué)修飾使一部分核糖上的2‘與4’碳連結(jié)在一起,增加了RNA的穩(wěn)定性。miRNA的過(guò)表達(dá)miRNA的功能研究miRNA
inhibitor是運(yùn)用化學(xué)方法
的miRNA抑制劑,可通過(guò)與成熟miRNA分子特異性結(jié)合而抑制miRNA作用,可以削弱細(xì)胞中內(nèi)源性miRNA導(dǎo)致的
調(diào)控作用,進(jìn)行miRNA功能缺失性(loss-of-function)研究。miRNA的表達(dá)抑制目的光素酶+熒的pGL4質(zhì)粒海腎熒光素酶基因的phRL質(zhì)粒Small
interfering
RNAssiRNA的發(fā)現(xiàn)1990年,Rich
Jorgensen將強(qiáng)啟動(dòng)子控制的Chalconesynthasegene轉(zhuǎn)入淡紫色的矮牽牛花,希望加深紫色。結(jié)果許多花出現(xiàn)雜色,甚至紫色共抑制:外源導(dǎo)入 和內(nèi)源。具有相似的 序列,導(dǎo)致內(nèi)源的表達(dá)受到抑制。后來(lái)在多種植物及真菌中都發(fā)現(xiàn)了共抑制現(xiàn)象,但其原因令人困惑。1995年,Su
Guo和Kemphuse,做了反義RNA阻斷線蟲(chóng)
表達(dá)的實(shí)驗(yàn)。其利用反義RNA阻斷線蟲(chóng)的par-1
。并在對(duì)照組中給線蟲(chóng)注射正義RNA以期觀察到表達(dá)的增強(qiáng)。結(jié)果,正義和反義RNA都能夠有效地抑制的表達(dá)!他們認(rèn)為這可能是由于當(dāng)中有部分的反義RNA的污
染所致。雖然后來(lái)文章
在當(dāng)年的Cell上,但卻遺憾地錯(cuò)過(guò)了生命科學(xué)史上的一個(gè)重大發(fā)現(xiàn)Small
interfering
RNAsRNAi
in
C.
elegans1998年,F(xiàn)ire等將正義和反義RNA的混合物注射到秀麗線蟲(chóng)中,發(fā)現(xiàn)其對(duì)內(nèi)源的抑制效果比注射單鏈正義或反義RNA還要顯著,因而作出了雙鏈RNA是 沉默的誘因這一論斷;同時(shí),他們提出了RNA干擾這一概念。Silencing
of
a
GFP
reporter
in
C.
elegans
occurs
when
animals
feed
onbacteriaexpressing
GFP
dsRNA
(a)
but
not
in
animals
that
are
defective
for
RNAi
(b).Note
that
silencing
occurs
throughout
the
body
of
the
animal,
with
the
exception
of
afew
cells
in
the
tail
that
express
some
residualGFP.
The
signalis
lost
in
intestinal
cellsnear
the
tail
(arrowhead)
as
wellas
near
the
head(arrow).
Thelack
of
GFP-positiveembryos
in
a
(bracketedregion)
demonstrates
the
systemic
spread
and
inheritance
ofsilencing.RNAi的研究歷程Small
interfering
RNAsSmall
interfering
RNAsThe
two
categories
of
small
RNAs
had e
firmly
embedded
inour
view
of
thegene
regulatory
landscape:
miRNAs,
as
regulators
of
endogenousgenes,
and
siRNAs,as
defenders
of
genome
integrity
in
responseto
foreign
or
invasive
nucleic
acids
such
as es,
transposons,and
transgenes.Small
interfering
RNAsA
Diversity
of
siRNA
SourcesSeveral
different
categoriesof
transcripts
canadopt
dsRNA
structures
thatcanbeprocessedby
Dicer
intosiRNAs.Small
interfering
RNAsMechanisms
of
siRNA
SilencingDuring
canonical
RNAi,
siRISCrecognizes
a
perfectlycomplementarymRNA,
leading
to
Ago-catalyzed
mRNAcleavage
at
a
single
site
withintheduplex.
After
cleavage,
functionalsiRISC
is
regenerated,
whereas
themRNA
fragmentsare
furtherdegraded.sIn
some
cases,they
can
silenceby
miRNA-like
mechanisms
involvingtranslational
repression
andexonucleolytic
degradation,
thoughthe
frequency
with
which
naturalsiRNAs
use
these
pathways
is
notclear.Small
interfering
RNAsFinally,
siRISC
can
direct
heterochromatin
formation
byassociating
with
nascent
transcripts
and
RNA
polymerases(RNA
Pol
II
in
S.
pombe
and
RNA
Pol
IV/V
in
A.
thaliana).Inplants, engagement
leads
to
the
association
oractivation
of
a
DNA
methyltransferase
(DMT)
that
methylatesthe
DNA,
leading
to
heterochromatin
formation.Small
interfering
RNAssiRNA
amplification
by
RdRPIn
most
eukaryotesotherthaninsects
and
mammals,recognition
by
siRISC
inducesthesynthesis
of
secondary
dsRNAs
andsiRNAs
by
RdRP
enzymes.Thesecondary
dsRNAs
are
processed
byDicer
intosiRNAs,
which
add
to
thepool
of
siRISC.
In
nematodes,
many
of
the
secondary
siRNAs
arise
assingle-stranded,
unprimed
transcriptswith
5’-triphosphates
and
do
notrequire
Dicer
processing.Small
interfering
RNAsDeliver
ofsiRNA2002年,BrummelKamp等首次使用小鼠H1啟動(dòng)子構(gòu)建了小發(fā)卡(smallhairpin
RNA,shRNA)表達(dá)載體,并證實(shí)轉(zhuǎn)染該載體可有效地、特意地細(xì)胞內(nèi)目的 的表達(dá)。Small
interfering
RNAssiRNA
Gene
SilencerssiRNA
refers
to
small
interfering
or
shortinterfering
RNA.Requirestransfection
of
cells
using
a
lipid-based
transfection
reagentusefulfor
atransient
knock-down.Small
interfering
RNAsshRNA
Plasmid
Gene
SilencersshRNA
refers
to
small
hairpin
or
short
hairpin
RNA.Plasmids
encoding
shRNA
enter
the
cell
via
lipid-basedtransfection.shRNA
plasmidsare
capable
of
transient
or
stableinhibition
of gene
expression.Smallinterfering
RNAsshRNA
Lentiviral
ParticlesLentiviral
delivery
of
shRNAexpressionconstruct
for
stable
integration
andexpresionof
shRNA.Non-coding
RNAs
mediating
RNAi4.Long
non-coding
RNAsAlexander
Hüttenhofer,
et
al.
Trends
Genet.
2005
May;21(5):289-97.Genomic
space
for
the
discovery
of
novel
RNAs
in
different
speciesEstimated
sizes
of
RNAfractions
of
representativebacterial
or
eukaryalgenomes,
which
are
eitherprotein-coding
or
non-protein
coding,
are
given
aspercentages
of
the
total
sizeof
the
respective
genome.RNAs
in
GenomeEmerging
classes
oflncRNAsLong
non-coding
RNAs
(lncRNAs;shown
in
blue)
mediate
a rray
of
genomicand
cellular
functions
and
are
independently
transcribed
from:
intergenic
regions;in
antisense,
overlap ,intronic
and
bidirectional
orientations
to
protein-codinggenes
(black);
from
gene-regulatory
regions,including
gene
promoters,
enhancersand
untranslated
regions(UTRs);
and
from
specific
chromosomal
regions,
includingomeres
(arrows
indicate
direction
of
transcription).lncRNA
plays
an
importantrole
in
ES
cells
self-renewand
differentiation
processHow
do
theywork?Long
ncRNAsFour
principles
of
nucleic
acid
and
protein
interactionsRNA–protein
interactions,
(2)
DNA–RNA
hybridization-based
interactions,(3)
DNA–protein
interactions
and
(4)
RNA–RNAhybridization
basedinteractions.Long
ncRNAsModels
of
long
noncoding
RNA
(lncRNA)
mechanisms
of
actioncis-tetherOne
modelof
ncRNAsthathave
a
cis-functionby
remaining
tethered
to
their
site
oftranscription.
In
this
model,
RNA
polymerase(green)
transcribes
an
RNA
(red),
whichcanassociate
with
regulatory
proteins
(purple)
toaffect
neighbouring
regions,
as
proposed
forXIST.ncRNAs
can
promote
spatial
rearrangement
of
thesurrounding
chromatin
contextlncRNA
recruits
the
histone
H3K4–modifying
complex
MLL1by
binding
toWDR5, ing
this
complex
to
the
HOX
genelocusenhancerInteraction
with
mRNAGuideScaffoldtrans-regulationOne
model
for
ncRNA
trans-regulation.
In
thismodel
an
ncRNA
can
associate
with
DNA-binding
proteins
(blue)
and
regulatoryproteinsto
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