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Pre-mRNAsplicing

VirologyI:Howviruseswork

Week8,session5

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DiscoveryofmRNAsplicinginAdinfectedcells

Major late mRNA mixture

RNase T1m7G5'ppp5'AmCmU(CCCCUUU)G

The same 5'-terminalsequence on multiplemRNAs

Isolationof capped

oligonucleotides

A

B

Adenoviral genome

A CB

1 32

C

3'

5'

3'5'

3

2

1

Hexon

mRNA

A

B

m7Gppp

m7Gppp

m

7

Gppp

Loops

Tripartite leadersequence

Hexon

mRNAbody

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Splicingofpre-mRNA

G/AG/AUGGAG/A GGACNUGA A C/UC U

Branch pointsequence

Exon 1

Pre-mRNA

Frequency of occurrence (%) 70 60 80 ~100 ~100 95 70 80 80 90 80 ~100 80 80

1840

nucleotides

IntronA

B

60~100 ~100

5' splice site 3' splice sitePolypyrimidine

tract

(~15 bases) Exon 2

45

AG

G

A

A

A

CU

C

U

G

G

A

UG

U

O

Firsttransesterification

Secondtransesterification

Spliced exons

Excised lariat intron

Intron

O

O H

5'

5'

5'

3'

3'

5'3'3'

3'

3'

2'

5'2'

OPO

O

O P O

Exon 1

5' O5'

Exon 2

3'

O

O

O

OPO

P

A

5'

O

O

O

OH

PO

O

O

O P O

O

O

U

G

G

A

A

OH

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RNA-RNAinterac9onsduringsplicing

U5

U6

U2

Helix II

U5

3'

5'

5'

U5

U1

U1

?

Helix II

Helix I

AG

U4

3'

U5

OH

Helix I

5'

3'

U6

U6

U2

3'

5'

AG

U1

U4

A

A

A

Prespliceosome

snRNPs

AG

UG

snRNPs

Pre-mRNA

Mature mRNA

3'5'

UG

A

AG

UG

A

A AGC

G

A

A AGC

G

U2

U2

U6

A

A A

G

C

G

U2

3'

5'

AG

UG

U1

A

Spliceosome

UG

1

2

3

4

5

spliceosome=ribozyme

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Splicing=Valueadded

Alterna)vesplicingcreatesnewfunc)onalgenes

Codinginforma)onofasmallDNAgenomeisexpanded

Regula)onofgeneexpression

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Cons9tu9veandalterna9vesplicing

Constitutive splicing gnicilpsevitanretlAA

Exon skipping Alternative 5' splice sites

B

5' 3' 5' 3'2 31

1 32

5' 3' 5' 3'

5'a

5'a

5'b

2 31 2

2

1 1'

1+ 1'

1+1'1 32 1 3 1 2

Alternative 3' splice sites

3'a

2'5'

3'a 3'b

3'b

21 2'

2'+2

2+2'1 21

5'b

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Alterna9vepolyadenyla9onandsplicing

B

C

D

mRNA3225

4203

mRNA3605 3764

71755609

DNA

7946/1 1000 3000 5000 7000

ORFs

E6

E7

E8

E1

E2

E4

E3 E5

L2 L1

PL

AE

AL

U1 snRNP

Pre-mRNA

L1 mRNA

Poly(A) polymerase

AL

7321Pseudo-5'splice site

A

Pre-mRNA

32253' splice site 3605

3' splice site3764

5' splice site

A

Splicing

Polyadenylation

SE1 ESS1 ESS2SE2 SE4

Yb-1SR/Asf SR/Asf

U2af,Asf,SR

U1-70k

early

3225 3605

granular

celllayer

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ControlofmRNAprocessing

duringretrovirusinfec9on

mRNA

Pre-mRNAmRNA

Gag Pol

Env

A

Unspliced:splicedRNA

Viralpropagation

2:1

1:1

1:2

Wild type

Reduced

Undetectable

Poly(A) signal

Pre-mRNA

5' splice

5' c

U1 snRNP

NRS

SR proteins

(A)n3'

Avianleukosisvirus,ALV

(negaEveregulatorysequence)

(decoy5splicesite)

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Alterna9vepolyadenyla9onandsplicing

duringadenovirusinfec9on

3'5' c

c

c

c

c

Poly(A) addition at one of five possible sites, L1 to L5

Pre-mRNA

Late

Major late promoter

AdenoviralDNA

0 16.5 pam00105 units

c

L1 L2 L3 L4 L5

L1

l1 l2 l3

L2 L3 L4 L5

An

An

52/55 kDa

IIIa

L1 mRNAs

Tripartite leadersequence

An

An

An

An

An

L3 mRNAs

An

RNA splicing

Protease

Two possible 3'splice sites

Alternative splicing

3L2L1L1L

4 possible 3'splice sites

c

c

c

c

c

c

c

c

pIV

Hexon

Transcription

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Viralproteinscanregulatealterna9vesplicing

c

cEarly

52/55-kDamRNA

Primary transcript

L1

L1c

SR

c

cLate

IIIa mRNA

L1

L4 33K

E4 orf4SR

+

c

3' splice sites

33K

P

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SplicingmarksmRNAsfornuclearexport

Export

Splicing

(A)n

3'5' c

(A)n 3'5' c

Ref/Aly (or SR proteins)

Uap56

Uap56

Tap

Cytoplasm

Nucleus

(A)n

3'5' c

Nxt1

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RevproteinregulatesexportofHIVmRNA

DNA

3'5' c

3'5' c

3'5' c

Splicing

RRE

Rev

Splicing

Transcription

Gag, Pol

Env, Vif, Vpu, Vpr

Rev

Rev

+ Rev

9-kb class

Nucleus

4-kb class

2-kb class

Cytoplasm

TatNef

Rev

gag

LTRLTR

HostHost pol

env

vpr

tat

rev nef

vif

vpu

5' c

5' c5' c

5' c

5' c

5' c

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RetroviralmRNAexportwithoutrev

ANRm-erpralulleCANRlarivorterdecilpsnU

Export Export

Splicing

(A)n

3'5' c

(A)n

3'5' c

(A)n

3'

CTEEnv

Ref/Aly (or SR proteins)

Uap56

Uap56

Refs

Tap

5' c

(A)n

3'5' c

Cytoplasm

Nucleus

(A)n

3'(A)n

3' 5' c

Tap

5' c

Tap

Nxt1

Nxt1Nxt1

ConsEtuEvetransportelement

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Table 10.3 Viral proteins that regulate RNA-processing reactions

Virus Protein(s) Functions

Adenovirus

Human adenovirus type 2 E4 ORF4 Induces dephosphorylation of cellular SR proteins by protein phosphatase

2A; relieves inhibition of L1 pre-mRNA splicing at the IIIa 3 splice site

by phosphorylated SR proteins present early in infection

E1B 55-kDaE4ORF6

The complex inhibits export of fully processed cellular mRNAs and inducesselective export of viral late mRNAs

L4 33 kDa Promotes alternative splicing to produce L1 IIIa mRNA

Herpesvirus

Herpes simplex virus type 1 ICP27 Stimulates polyadenylation of viral late mRNAs with suboptimal sequences

at their polyadenylation sites; inhibits splicing of intron-containing

cellular and probably viral mRNAs; promotes export of viral single-exon

mRNAs

RetrovirusHuman immunodeficiency virus type 1 Rev Mediates export of unspliced and incompletely spliced viral mRNAs

NT-08-0601-C010.indd 362 9/8/08

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W008 S005 Virology