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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