讲稿表观遗传学-non-cordingrna p

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分三类：5SrRNA、16SrRNA和23SrRNA。真核生物的rRNA分四类：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表观遗传学课上的两类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

5ʹ

strand

and

onefrom

the

3ʹ

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

pocket

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

5ʹ-to-3ʹ

exoribonuclease

XRN4

in

plants

and

XRN1

inanimals,

together

with

the

major

cellular

3ʹ-to-5ʹ

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

5ʹ-to-3ʹ

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的表达检测miRNAmiRNAReal

timeQ-PCR检测miRNA

发夹前体序列已升至24521

条，新增3

千余条。成熟miRNA

序列升至30424

条，新增5

千余条。人成小鼠成miRNA

新增至2578

条；miRNA

新增至1908

条；大鼠miRNA

新增至728

条。miRNA

mimic是运用化学方法

的双链RNA，能模拟细胞中内源性成熟miRNA的高水平表达，以增强内源性miRNA的调控作用，进行功能获得性(gain-of-function)研究。只需直接转染进入细胞，即可检测功能变化，快速，方便。LNA（Locked

Nucleic

Acid），通过化学修饰使一部分核糖上的2‘与4’碳连结在一起，增加了RNA的稳定性。miRNA的过表达miRNA的功能研究miRNA

inhibitor是运用化学方法

的miRNA抑制剂，可通过与成熟miRNA分子特异性结合而抑制miRNA作用，可以削弱细胞中内源性miRNA导致的

调控作用，进行miRNA功能缺失性(loss-of-function)研究。miRNA的表达抑制目的光素酶+荧的pGL4质粒海肾荧光素酶基因的phRL质粒Small

interfering

RNAssiRNA的发现1990年，Rich

Jorgensen将强启动子控制的Chalconesynthasegene转入淡紫色的矮牵牛花，希望加深紫色。结果许多花出现杂色，甚至紫色共抑制：外源导入 和内源。具有相似的 序列，导致内源的表达受到抑制。后来在多种植物及真菌中都发现了共抑制现象，但其原因令人困惑。1995年，Su

Guo和Kemphuse，做了反义RNA阻断线虫

表达的实验。其利用反义RNA阻断线虫的par-1

。并在对照组中给线虫注射正义RNA以期观察到表达的增强。结果，正义和反义RNA都能够有效地抑制的表达！他们认为这可能是由于当中有部分的反义RNA的污

染所致。虽然后来文章

在当年的Cell上，但却遗憾地错过了生命科学史上的一个重大发现Small

interfering

RNAsRNAi

in

C.

elegans1998年，Fire等将正义和反义RNA的混合物注射到秀丽线虫中，发现其对内源的抑制效果比注射单链正义或反义RNA还要显著，因而作出了双链RNA是 沉默的诱因这一论断；同时，他们提出了RNA干扰这一概念。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的研究历程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启动子构建了小发卡（smallhairpin

RNA，shRNA）表达载体，并证实转染该载体可有效地、特意地细胞内目的 的表达。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