醫(yī)用分子遺傳學(xué)轉(zhuǎn)錄和轉(zhuǎn)錄后修飾課件

李

希分子醫(yī)學(xué)教育部重點實驗室lixi@Transcription

and

Post-transcriptionModification1Post-transcriptionalProcessing

of

RNA2Making

ends

of

RNARNA

splicing3Primary

TranscriptPrimary

Transcript-the

initial

molecule

of

RNAproduced---

hnRNA

（heterogenousnuclearRNA

）In

prokaryotes,

DNA

→RNA

→protein

incytoplasm

concurrentlyIn

eukaryotes

nuclear

RNA

>>

Cp

RNAProcessing

of

eukaryoticpre-mRNAHuman

dystrophin

gene

has

79exons,

spans

over

2,300-Kb

andrequires

over

16

hours

to

be4transcribed!For

primarytranscriptscontaining

multipleexons

and

introns,splicing

occurs

beforetranscription

of

the

geneis

complete--co-transcriptional

splicing.Types

of

RNA

processingCutting

and

trimming

to

generate

ends: rRNA,

tRNA

and

mRNACovalent

modification:Add

a

cap

and

a

polyA

tail

to

mRNAAdd

a

methyl

group

to2’-OH

of

ribose

in

mRNA

andrRNAExtensive

changes

of

bases

in

tRNASplicingpre-rRNA,

pre-mRNA,

pre-tRNA

by

differentmechanisms.5The

RNA

Pol

II

CTD

is

required

for

the

coupling

of

transcription

withmRNA

capping,

polyadenylation

and

splicingThe

coupling

allowstheprocessingfactors

to

present

athighlocalconcentrations

whensplice

sites

andpoly(A)

signals

aretranscribed

by

Pol

II,enhancing

the

rateand

specificity

ofRNA

processing.The

association

ofsplicing

factors

withphosphorylated

CTDalso

stimulates

PolIIelongation.

Thus,

apre-mRNA

is

notsynthesized

unlessthe

machineryforprocessing

it

isproperly

position6ed.Time

course

of

RNA

processing75’

and

3’

ends

of

eukaryotic

mRNAAdd

a

GMPMethylate

it

and1st

few

nucleotidesCut

the

pre-mRNAand

add

A’s5’-UTR3’-UTR89Capping

ofpre-mRNAsCap=modified

guanine

nucleotideCapping=

first

mRNA

processing

event

-

occurs

duringtranscriptionCTD

recruits

capping

enzyme

as

soon

as

it

isphosphorylatedPre-mRNAmodified

with

7-methyl-guanosinetriphosphate

(cap)

when

RNA

is

only25-30

bp

longCapstructureisrecognizedby

CBC（cap-bindingcomplex）stablize

the

transcriptprevent

degradation

by

exonucleasesstimulate

splicing

and

processingSometimesmethylatedSometimesmethylatedThe

cap

is

added

afterthe

nascent

RNAmolecules

produced

byRNA

polymerase

IIreach

a

length

of

25-30nucleotides.Guanylyltransferase

isrecruited

and

activatedthrough

binding

to

theSer5-phosphorylatedPol

II

CTD.The

methyl

groupsare

derived

from

S-adenosylmethionine.Capping

helpsstabilize

mRNA

andenhances

translation,splicing

and

exportinto

the

cytopl1a0sm.Capping

of

the

5’

end

of

nascent

RNA

transcripts

with

m7GExistingina

singlecomplexConsensus

sequence

for

3’

processAAUAAA:

CstF

(cleavage

stimulation

factorF)GU-rich

sequence:

CPSF

(cleavage

andpolyadenylationspecificity

factor)11Polyadenylation

of

mRNA

at

the

3’

endCPSF:

cleavage

and

polyadenylation

specificityfactor.CStF:

cleavage

stimulatory

factor.CFI

&

CFII:

cleavage

factor

I

&

II.PAP:

poly(A)

polymerase.PABPII:poly(A)-binding

protein

II.RNA

is

cleaved

10~35-nt

3’

to

A2UA3.The

binding

of

PAP

prior

to

cleavage

ensuresthat

the

free

3’

end

generated

israpidlypolyadenylated.PAP

adds

the

first

12A

residues

to

3’-OHslowly.Binding

of

PABPII

to

the

initial

shortpoly(A)

tail

accelerates

polyadenylation

byPAP.Poly(A)

tail

stabilizes

mRNA

andenhancestranslation

and

export

into

thecytoplasm.The

polyadenylation

complex

is

associatedwiththe

CTD

of

Pol

II

followinginitiation.1213Functions

of

5’

cap

and

3’

polyANeed

5’

cap

for

efficient

translation:Eukaryotic

translation

initiation

factor

4

(eIF4)recognizes

and

binds

to

the

cap

as

part

ofinitiation.Both

cap

and

polyA

contribute

to

stability

of

mRNA:Most

mRNAs

without

a

cap

or

polyA

aredegradedrapidly.Shortening

of

the

polyA

tail

and

decapping

are

part

ofone

pathway

for

RNA

degradation

in

yeast.14mRNA

Half-lifet?

seconds

if

seldom

neededt?

several

cell

generations

(i.e.~48-72

h)

for houskeeping

gene≈avg

3

h

ineukaryotes≈avg

1.5

min

inbacteriaPoly(A)+

RNA

can

be

separated

from

other

RNAsby

fractionation

on

Sepharose-oligo(dT).1516Split

gene

and

mRNA

splicingBackground: Adenovirus

has

a

DNA

genome

andmakes

many

mRNAs. Can

we

determine

whichpart

of

the

genome

encodes

for

each

mRNA

bymaking

a

DNA:RNA

hybrid?Experiment: Isolate

Adenovirus

genomic

DNA,isolate

one

adenovirus

mRNA,

hybridize

andthen

look

by

EM

at

where

the

RNA

hybridizes(binds)

to

the

genomic

DNA.Surprise: The

RNA

is

generated

from

4different

regions

of

the

DNA! How

can

weexplain

this?

Splicing!!17The

discovery

of

split

genes

(1977)1993

Noble

Prize

in

MedicineTo

Dr.

Richard

Robert

and

Dr.

Phillip

SharpThe

matured

mRNAs

are

much

shorterthanthe

DNAtemplates.DNAmRNA1819Exon

andIntron

Exon

is

any

segment

of

an

interrupted

genethat

is

represented

in

the

mature

RNAproduct.

Intron

is

a

segment

of

DNA

that

istranscribed,

but

removed

from

within

thetranscript

by

splicing

together

thesequences

(exons)

on

either

side

of

it.Exons

aresimilar

in

size20Introns

arehighlyvariable

in

sizeGT-AG

ruleGT-AG

rule

describes

the

presence

of

these

constant

dinucleotides

at

the

firsttwo

and

last

two

positions

of

introns

of

nuclear

genes.Splice

sites

are

the

sequences

immediately

surrounding

the

exon-intron

boundariesSplicing

junctions

are

recognized

only

in

the

correct

pairwise

combinations21The

sequence

of

steps

in

the

production

of

mature

eukaryotic

mRNA

asshownfor

the

chicken

ovalbumin

gene.The

consensus

sequence

at

the

exon–intron

junctions

of

vertebrate

pre-mRNAs.2223244

major

types

ofintrons4

classes

of

introns

can

be

distinguished

on

the

basisof

their

mechanism

of

splicing

and/orcharacterisiticsequences:Group

I

introns

in

fungal

mitochondria,

plastids,andinpre-rRNA

inTetrahymena(self-splicing)Group

II

introns

in

fungal

mitochondria

andplastids

(self-splicing)Introns

inpre-mRNA

(spliceosome

mediated)Introns

in

pre-tRNAGroup

I

and

II

introns25The

sequence

of

transesterification

reactions

thatsplice

together

the

exons

of

eukaryotic

pre-mRNAs.2627Splicing

of

Group

I

and

II

intronsIntrons

in

fungal

mitochondria,

plastids,

Tetrahymena

pre-rRNAGroup

ISelf-splicingInitiate

splicing

with

aG

nucleotideUses

a

phosphoester

transfer

mechanismDoes

not

require

ATPhydrolysis.Group

IIself-splicingInitiate

splicing

with

an

internal

AUses

a

phosphoester

transfer

mechanismDoes

not

require

ATPhydrolysisSelf-splicing

in

pre-rRNA

in

Tetrahymena

:T.

Cech

et

al.

1981pre-rRNASpliced

exonIntron

circleIntronlinear

pre-rRNANuclear

extractGTP28+

+

+

+-

+

-

+-

+

+

-+Exon

1 Intron

1 Exon

2 Exon

1 Exon

2 Intron

1Products

of

splicing

were

resolved

by

gel

electrophoresis:Additional

proteinsare

NOT

needed

forsplicing

of

thispre-rRNA!Do

need

a

Gnucleotide

(GMP,GDP,

GTP

orGuanosine).The

sequence

of

reactions

in

the

self-splicing

ofTetrahymena

group

I

intron.2930Where

is

the

catalytic

activity

in

RNase

P?RNase

P

is

composed

of

a

375

nucleotide

RNA

anda

20

kDa

protein.

The

protein

component

will

NOTcatalyze

cleavage

on

its

own.The

RNA

WILL

catalyze

cleavage

by

itself

!!!!The

protein

component

aids

in

the

reaction

but

is

notrequired

forcatalysis.Thus

RNA

can

be

anenzyme.Enzymes

composed

of

RNA

are

called

ribozymes.Hammerhead

ribozymes31A

58

nt

structure

is

used

in

self-cleavageThe

sequence

CUGA

adjacent

to

stem-loops

is

sufficient

for

cleavage32Mechanism

of

hammerhead

ribozymeThe

folded

RNA

forms

an

active

site

forbinding

a

metal

hydroxideAttracts

a

proton

from

the

2’

OH

of

thenucleotide

at

the

cleavage

site.This

is

now

a

nucleophile

for

attack

on

the

3’phosphate

and

cleavage

of

the

phosphodiesterbond.1989

Nobel

Prize

in

chemistry,

Sidney

Altman,and

ThomasCech33Distribution

of

Group

I

intronsProkaryotes

–

eubacteria

(tRNA

&

rRNA),

phageEukaryoteslower

(algae,

protists,

&

fungi)nuclear

rRNA

genes,

organellar

genes,

Chlorellaviruseshigher

plants:

organellar

geneslower

animals

(Anthozoans):

mitochondrial>1800

known, classified

into

~12

subgroups, basedonsecondary

structure34Splicing

of

pre-mRNAThe

introns

begin

and

end

with

almost

invariantsequences:

5’

GU…AG

3’Use

ATP

hydrolysis

to

assemble

a

largespliceosome

(45S

particle,

5

snRNAs

and

65proteins,

same

size

and

complexity

as

ribosome)Mechanism

is

similar

to

that

of

the

Group

IIfungalintrons:Initiate

splicing

with

an

internal

AUses

a

phosphoester

transfer

mechanism

forsplicingInitiation

of

phosphoester

transfers

in

pre-mRNAUses 2’

OH

of

an

A

internal

to

theintronForms

a

branch

point

by

attackingthe

5’

phosphate

on

the

firstnucleotide

of

the

intronForms

a

lariat

structure

in

theintronExons

are

joined

and

intron

isexcised

as

a

lariatA

debranching

enzyme

cleaves

thelariat

at

the

branch

to

generate

alinear

intronLinear

intron

is

degraded3536Involvement

of

snRNAs

and

snRNPssnRNAs

=

small

nuclear

RNAssnRNPs

=

small

nuclearribonucleoproteinsparticles

(snRNA

complex

with

protein)Addition

of

these

antibodies

to

an

in

vitro

pre-mRNA

splicing

reactionblocked

splicing.Thus

the

snRNPs

were

implicated

in

splicing37Recognizing

the

5’

splice

site

and

the

branch

site.Bringing

those

sites

together.Catalyzing

(or

helping

to

catalyze)

the

RNA

cleavage.Role

of

snRNPsin

RNA

splicingRNA-RNA,RNA-protein

and

protein-proteininteractions

are

all

important

during

splicing38snRNPsU1,

U2,

U4/U6,

and

U5

snRNPsHave

snRNA

in

each:

U1,

U2,

U4/U6,

U5Conserved

from

yeast

to

humanAssemble

into

spliceosomeCatalyze

splicingSplicing

of

pre-mRNA

occurs

in

a“spliceosome”

an

RNA-protein

complexpre-mRNA spliced

mRNAThe

spliceosome

is

a

largeprotein-RNAcomplexin

which

splicing

of

pre-mRNAs

occurs.39spliceosome(~100

proteins

+

5

smallRNAs)40Assembly

of

spliceosomesnRNPs

are

assembled

progressively

into

thespliceosome.U1

snRNP

binds

(and

base

pairs)

to

the

5’

splice

siteBBP

(branch-point

binding

protein)

binds

to

the

branchsiteU2

snRNP

binds

(and

base

pairs)

to

the

branch

point,

BBPdissociatesU4U5U6

snRNP

binds,

and

U1

snRNP

dissociatesU4

snRNP

dissociatesAssembly

requires

ATP

hydrolysisAssembly

is

aided

by

various

auxiliary

factors

andsplicing

factors.Some

RNA-RNA

hybrids

formedduring

the

splicing

reactionSteps

of

thespliceosome-mediated

splicing

reaction41A

schematic

diagram

of

six

rearrangements

that

the

spliceosome

undergoes

inmediating

the

first

transesterification

reaction

in

pre-mRNA

splicing.Assembly

of

spliceosome42The

spliceosomecycle4344The

Significance

of

Gene

SplicingThe

introns

are

rare

in

prokaryotic

structuralgenesThe

introns

are

uncommon

in

lower

eukaryote(yeast),

239

introns

in

~6000

genes,

only

oneintron

/polypeptideThe

introns

are

abundant

in

higher

eukaryotes(lacking

introns

are

histons

and

interferons)Unexpressed

sequences

constitute

~80%

of

atypical

vertebrate

structural

geneErrors

produced

by

mistakesin

splice-siteselection45Mechanisms

prevent

splicing

errorCo-transcriptional

loading

processSR

proteins

recruit

spliceosome

components

to

the

5’

and3’

splice

sitesSR

protein

=

Serine

Arginine

rich

proteinESE

=

exonic

splicingenhancersSR

protein

regulates

alternative

splicing46Alternative

splicingAlternative

splicing

occurs

in

all

metazoa

and

isespecially

prevalent

in

vertebrateFive

ways

to

splice

an

RNA47Regulated

alternative

splicingDifferent

signalsinthe

pre-mRNA

anddifferent

proteinscause

spliceosomesto

form

inparticularpositions

togivealternative

splicing4856775567Fas

pre-mRNAAPOPTOSIS

(programmedcell

death)(-)Alternative

splicing

can

generate

mRNAs

encodingproteins

with

different,

even

opposite

functionsFas

ligandSoluble

Fas(membrane)Fas

ligandFas49(membrane-associated)(+)Alternative

possibilities

for

4

exons

leave

a

total

number

of

possiblemRNA

variations

at

38,016.

The

protein

variants

are

important

forwiring

of

the

nervous

system

and

for

immune

response.50Drosophila

Dscam

gene

contains

thousandsof

possible

splice

variantsCis-and

Trans-SplicingCis-: Splicing

in

single

RNATrans-:

Splicing

in

two

different

RNAsY-shaped

excised

introns

(cis-:lariat)Occur

in

C.

elegance

and

higher

eukaryotes

but

it

does

in

only51a

few

mRNAs

and

at

a

very

low

levelpre-mRNAsplicing

trans-mRNA

splicingsplicedleaderSame

splicing

mechanism

isemployed

in

trans-splicing52Spliced

leader

contains

the

cap

structure!53RNA

editingRNA

editing

is

the

process

of

changing

thesequence

of

RNA

after

transcription.In

some

RNAs,

as

much

as

55%

of

thenucleotidesequence

is

not

encoded

in

the

(primary)

gene,

butis

added

after

transcription.Examples:

mitochondrial

genes

inTrypanosomes（錐蟲）Can

add,

delete

or

change

nucleotides

by

editing54Two

mechanisms

mediateeditingGuide

RNA-directeduridine

insertionordeletionSite-specific

deaminationInsertion

and

deletion

of

nucleotides

by

editingUses

a

guide

RNA(in

20S

RNP

=editosome)

that

isencoded

elsewherein

thegenomePart

of

the

guideRNA

iscomplementary

tothe

mRNA

invicinityof

editingTrypanosomal

RNA

editing

pathways.(a)

Insertion.

(b)

Deletion.55Mammalian

example

of

editingThe

C

is

converted

to

U

in

intestine

by

aspecificdeaminating

enzyme,

not

by

a

guideRNA.5657Cutting

and

Trimming

RNACan

use

endonucleases

to

cut

at

specific

siteswithin

a

longer

precursorRNACan

use

exonucleases

to

trim

back

from

thenew

ends

to

make

the

matureproductThis

general

process

is

seen

inprokaryotes

andeukaryotes

for

all

types

of

RNAThe

posttranscriptional

processing

ofE.

colirRNA.58RNase

III

cuts

in

stems

of

stem-loops16S

rRNA5923S

rRNARNaseIIINo

apparent

primary

sequence

specificity

-

perhaps

RNase

IIIrecognizes

a

particular

stem

structure.60Eukaryotic

rRNA

ProcessingThe

primary

rRNA

transcript

(~7500nt,

45SRNA)contains

18S,

5.8S

and

28SMethylationoccur

mostly

in

rRNAsequence80%:

O2-methylribose,

20%:

bases

(A

or

G)peudouridine95

U

in

rRNA

in

human

are

converted

to

’smay

contribute

rRNA

tertiary

stabilityTransfer

RNA

ProcessingCloverleaf

structureCCA:

amino

acid

bindingsiteAnticodon~60

tRNA

genes

in

E.coliA

schematic

diagram

of

thetRNAcloverleaf

secondary

structur6e1.Endo-

and

exonucleases

to

generateends

oftRNAEndonuclease

RNase

P

cleaves

to

generate

the

5’end.Endonuclease

RNase

F

cleaves

3

nucleotides

past

the

mature

3’end.Exonuclease

RNase

D

trims

3’

to

5’,

leaving

the

mature

3’

end.6263Splicing

of

pre-tRNAIntrons

inpre-tRNA

are

very

short

(about

10-20nucleotides)Have

no

consensussequencesAre

removed

by

a

series

of

enzymaticsteps:Cleavage

by

an

endonucleasePhosphodiesterase

to

open

a

cyclic

intermediate

andprovide

a3’OHActivation

of

one

end

by

a

kinase

(with

ATPhydrolysis)Ligation

of

the

ends

(with

ATP

hydrolysis)Phosphatase

to

remove

the

extra

phosphate

on

the2’OH

(remaining

after

phosphodiesterase

)Steps

in

splicing

ofpre-tRNAP2’,3’

cyclicphosphateOH

5’Excised

intronIntron

of10-20nucleotides1.Endo-nucleasePhospho-diesteraseKinase

(ATP)Ligase

(ATP)Phosphatase++64SplicedtRNA65CCA

at

3’

end

of

tRNAsAll

tRNAs

end

in

the

sequence

CCA.Amino

acids

are

added

to

the

CCA

end

during“charging”

of

tRNAs

fortranslation.For

most

eukaryotic

tRNAs,

the

CCA

is

addedafter

transcription,

in

a

reaction

catalyzed

by

tRNAnucleotidyl

transferase.All

of

the

four

bases

in

tRNA

can

bemodified6667Pathologies

resulting

from

aberrant

splicingcan

be

grouped

in

two

major

categoriesMutations

affecting

proteins

that

are

involved

in

splicingExamples:Spinal

Muscular

AtrophyRetinitis

PigmentosaMyotonic

DystrophyMutations

affecting

a

specific

messenger

RNA

and

disturbing

itsnormal

splicing

patternExamples:β-ThalassemiaDuchenne

Muscular

DystrophyCystic

FibrosisFrasierSyndromeFrontotemporal

Dementia

and

Parkinsonism68Intron

Advantage?One

benefit

of

genes

with

introns

is

a

phenomenon

calledalternative

splicingA

pre-mRNA

with

multiple

introns

can

be

spliced

indifferentwaysThis

will

generate

mature

mRNAs

with

differentcombinations

of

exonsThis

variation

in

splicing

can

occur

in

different

cell

types

orduring

different

stages

of

development69Intron

Advantage?The

biological

advantage

of

alternative

splicingis

that

two

(or

more)

polypeptides

can