Lo splicing dell’RNA
• definizione
• importanza
• predizione
Ing Francesco Piva
Gruppo di biologia computazionale e molecolare
Dipartimento di Biochimica, Biologia e Genetica
Università Politecnica delle Marche
Edificio Scienze 3, Brecce Bianche, Ancona
[email protected]
These are synonymous words, are we sure ?
. . . Ala Val
Arg . . .
GCA GTA CGA
C
C
C
G
G
G
T
T
T
AGA
G
4 * 4 * 6 = 96
Three AAs specified by 96
synonymous words
GCAGTACGA
GCAGTACGC
GCAGTACGG
GCAGTACGT
GCAGTAAGA
GCAGTAAGG
GCAGTCCGA
GCAGTCCGC
GCAGTCCGG
GCAGTCCGT
GCAGTCAGA
GCAGTCAGG
GCAGTGCGA
GCAGTGCGC
GCAGTGCGG
GCAGTGCGT
GCAGTGAGA
GCAGTGAGG
GCAGTTCGA
GCAGTTCGC
GCAGTTCGG
GCAGTTCGT
GCAGTTAGA
GCAGTTAGG
GCCGTACGA
GCCGTACGC
GCCGTACGG
GCCGTACGT
GCCGTAAGA
GCCGTAAGG
GCCGTCCGA
GCCGTCCGC
GCCGTCCGG
GCCGTCCGT
GCCGTCAGA
GCCGTCAGG
GCCGTGCGA
GCCGTGCGC
GCCGTGCGG
GCCGTGCGT
GCCGTGAGA
GCCGTGAGG
GCCGTTCGA
GCCGTTCGC
GCCGTTCGG
GCCGTTCGT
GCCGTTAGA
GCCGTTAGG
GCGGTACGA
GCGGTACGC
GCGGTACGG
GCGGTACGT
GCGGTAAGA
GCGGTAAGG
GCGGTCCGA
GCGGTCCGC
GCGGTCCGG
GCGGTCCGT
GCGGTCAGA
GCGGTCAGG
GCGGTGCGA
GCGGTGCGC
GCGGTGCGG
GCGGTGCGT
GCGGTGAGA
GCGGTGAGG
GCGGTTCGA
GCGGTTCGC
GCGGTTCGG
GCGGTTCGT
GCGGTTAGA
GCGGTTAGG
GCTGTACGA
GCTGTACGC
GCTGTACGG
GCTGTACGT
GCTGTAAGA
GCTGTAAGG
GCTGTCCGA
GCTGTCCGC
GCTGTCCGG
GCTGTCCGT
GCTGTCAGA
GCTGTCAGG
GCTGTGCGA
GCTGTGCGC
GCTGTGCGG
GCTGTGCGT
GCTGTGAGA
GCTGTGAGG
GCTGTTCGA
GCTGTTCGC
GCTGTTCGG
GCTGTTCGT
GCTGTTAGA
GCTGTTAGG
Genomic DNA
ATGTAAACGTATATCGTGACAGTGGTCTGTTAGTATTCCTTTAGTCATGGTTT
ATGTAAACTGGTCTGTTATCATGGTTT
mRNA
attggaaaccgaaacccgttggtcacctctgcaatagccctccctccctcacttctacaattttgtgacagtggtctt
gttttctgcattctctgcttcacgtgcttgttttgttggagcgcgtttgcatgctgctttaaattctgaaatattaaa
aaaatttcgaagtttttcagcacatgggatgggagttttgaatttcaattttttaaaaacatttttctgtgattagtg
ccgtcgtggcacggctgttagccgcctatccggtttattcgatactttGTGAGTTTTTTGTAACTTTATGGTCGTCGA
AATGGGAAAACTTGGCCACCAATATAAGTTTGGAAAACAATTTCCTAAAAATAAAATAATTGAACTTTTCCGATGAAT
AAAAAAATCGATCAGATATTCTGGAAAAAAAATCGATAAATTAATCGATTTTCTTGGAAAATACATCGAAAAATTGAG
AAAAATAGAAAAATGAATGTTTTTCGATTACCGATTTATTGATTTTTCGTGAAAACTGAGTTCAGATAATTTTAAAAG
CAATGTTTTTCATTTTTCAAATCAGAATCACTATAGTTTTGAAAAATCAATAATTAATTTATTGATTTTTCAATATAA
TTTTTTGGAAAAAATAGAAAAATCCCTTTCTAAAAGTTTTAAATTTCCAAGAAAAATTCATTTTCAAAATCACCAACG
CGCTCTATAGAGTAGTCGATGAAAATCTCCGTTAAGGGTGCATGGGCAAAACGCGCTCGAACGACAATTGTTATTGTA
TGTTTGGTCTTGCAACGAAAAGTTTGAAAAATTGAAAAAAAGTTGTGTCTGATACATTTTTTTTTGGCATTTTCTGCT
ATTTTACACCAGAAAAAATTTAATAAACATAAAAAATCGAAATTTTTCAAGTTGGACAATTTTCAGtgagcatcttat
ccatcctagttctcagttcaggacttgtgcacattcgtttagagccagatattcgcaaagccttttcaccggatgatt
cagatgctggataGTAAGTGACTACTGACCTTGAAGCCTCCTTCCTCCACCAGTCAGAAATAACACGTTTTTTCGCAA
TGTTTTTCTTTTTCTAATTCGATTTCCCTTTCTCCCTTTCTTATTGTGATTTGGTCAATGTTTGGTTGACTGGGAAGA
AAATTGAATTTTTTTGGAATTCCACTTGAAGTTAAAAAACCCAAAATAAATATTTGATCAAAAATAAATAAGAAAAAA
AAGAAAACTTTAAAGCAAATGAAAATTTCGTTCGTAACTATTTTGTTAATTTTTTTAAAACTCCTATTTTAAATATAT
GCTTTTTGCGGAAATTTCTATAAATTTTTTTACATTTTTCAGtgaaacccgtgtctggctggaatactacggactcga
catctatccggaacgagcattctgtatttttaccgccaagcgcgaaaattccagtattctccaggaaggcgcactggc
agacGTAAGTTGATTCTCCGTCACGCCCACTTTTCTGGCGGGAATTTAAAAAATTTCAGatttatactgtggacaatc
gactatcggcggcagttggctaccaagatggggatggacgaaaaaattgcgatccactctgcgacttgaacagcccct
ttcacttgttagcgGTAGGTGGTGGTCTAGGGTGTCATTTTTCGATTTTTTCAATTATTCGATGTTTTTAGTGAAAAT
CGAAAAATCTAAAAATTGAAAATCGAAAAATGAAAGAAACATTGTTTTTTGGGGACCAAACATCTTAATGAATTTAAC
AACAGGGAAAACTGAACAGAAACCTGGACGGTCTTATCCCATTTATCTATATTCTTAAAATGAATGATGGAGAAAAAA
GTTAAAATAAAAACATTATCAGCTTTTTGTAAGTTTTTCTCAAAAATTGTTCGATTTTTCGATTTTCTAAAAAGTCGA
AAAACCGAAACCCTTGGTGGTGGTGGTGGTGGACTAGAAAACTCTTCAACGACCACATGGCAATTTTCAGaatttgac
gcggagaaacaatggtaccacaagtgtattcacctatccggatatgccatatagcggactggatattttcctgggact
tcacttgagtaatgcggattttggtaagattttttttgaaatgttaaatgaaaagttgaaaaatagtttttatgattt
agccactttccagttaaaatttcatttttttaactataaaaagttctggaaaaatg
Struttura tipica dei geni umani
esoni
introni
esone1
introne1
GT
S
P
L
I
C
I
N
G
esone2
AG
introne2
GT
esone3
AG
eliminazione introni
introne1
introne2
esone2
esone1
esone3
unione esoni
esone1
esone2
esone3
Lo splicing avviene in tutto il trascritto, anche nelle zone non codificanti
R = G, A
Y = T, C
Meccanismo di splicing
estere
alcool
O
R
C
ORI
+
HO
RII
+
HO
RI
=
O
R
C
ORII
due legami fosfoesterici
U2AF si lega al tratto
pirimidinico a valle del
sito di ramificazione
Arg-Ser
arly
snRNP U2 si lega al
sito di ramificazione
(richiesta idrolisi ATP)
U2AF
U2AF
il 3’ss è tagliato e gli esoni
vengono saldati insieme, il
cappio verrà deramificato
le prot SR connettono
U2Af con snRNP U1
U2AF
snRNP U4 è rilasciato (richiesta idrolisi
ATP), snRNP U6 e U2 catalizzano la
transesterificazione, snRNP U5 si lega al
3’ss, il 5’ ss è tagliato e si forma il cappio
U2AF
si
legano
insieme
snRNP U5 si lega al 5’ss,
snRNP U6 si lega a snRNP U2
snRNP U1 è rilasciato, snRNP U5
si sposta dall’esone all’introne,
snRNP U6 si lega al 5’ss
introne (5’ss)
snRNP U1
Sm protein
G16
C5
RBD: RNA binding
domain
snRNP U2
si appaiano
con snRNA U6
Sm protein
si appaia al sito di
ramificazione
U17
U5
Lo splicing è tessuto specifico
Muscolo cardiaco
1
1
2
Muscolo uterino
2
3
5
3
1
4
3
4
5
5
Esempio di alternative splicing di un gene umano
Alternative splicing tessuto specifico
Tutti i modi di fare
splicing alternativo
Alcuni genomi virali
subiscono splicing
all’interno della cellula
ospite
equine infectious anemia virus (EIAV)
AIM:
mRNA
structure
pre mRNA
sequence
SPLICING PREDICTION TOOL
attggaaaccgaaacccgttggtcacctctgcaatagccctccctccctcacttctacaattttgtgaca
gtggtcttgttttctgcattctctgcttcacgtgcttgttttgttggagcgcgtttgcatgctgctttaa
attctgaaatattaaaaaaatttcgaagtttttcagcacatgggatgggagttttgaatttcaatttttt
aaaaacatttttctgtgattagtgccgtcgtggcacggctgttagccgcctatccggtttattcgatact
ttGTGAGTTTTTTGTAACTTTATGGTCGTCGAAATGGGAAAACTTGGCCACCAATATAAGTTTGGAAAAC
AATTTCCTAAAAATAAAATAATTGAACTTTTCCGATGAATAAAAAAATCGATCAGATATTCTGGAAAAAA
AATCGATAAATTAATCGATTTTCTTGGAAAATACATCGAAAAATTGAGAAAAATAGAAAAATGAATGTTT
TTCGATTACCGATTTATTGATTTTTCGTGAAAACTGAGTTCAGATAATTTTAAAAGCAATGTTTTTCATT
TTTCAAATCAGAATCACTATAGTTTTGAAAAATCAATAATTAATTTATTGATTTTTCAATATAATTTTTT
GGAAAAAATAGAAAAATCCCTTTCTAAAAGTTTTAAATTTCCAAGAAAAATTCATTTTCAAAATCACCAA
CGCGCTCTATAGAGTAGTCGATGAAAATCTCCGTTAAGGGTGCATGGGCAAAACGCGCTCGAACGACAAT
TGTTATTGTATGTTTGGTCTTGCAACGAAAAGTTTGAAAAATTGAAAAAAAGTTGTGTCTGATACATTTT
TTTTTGGCATTTTCTGCTATTTTACACCAGAAAAAATTTAATAAACATAAAAAATCGAAATTTTTCAAGT
TGGACAATTTTCAGtgagcatcttatccatcctagttctcagttcaggacttgtgcacattcgtttagag
ccagatattcgcaaagccttttcaccggatgattcagatgctggataGTAAGTGACTACTGACCTTGAAG
CCTCCTTCCTCCACCAGTCAGAAATAACACGTTTTTTCGCAATGTTTTTCTTTTTCTAATTCGATTTCCC
TTTCTCCCTTTCTTATTGTGATTTGGTCAATGTTTGGTTGACTGGGAAGAAAATTGAATTTTTTTGGAAT
TCCACTTGAAGTTAAAAAACCCAAAATAAATATTTGATCAAAAATAAATAAGAAAAAAAAGAAAACTTTA
AAGCAAATGAAAATTTCGTTCGTAACTATTTTGTTAATTTTTTTAAAACTCCTATTTTAAATATATGCTT
TTTGCGGAAATTTCTATAAATTTTTTTACATTTTTCAGtgaaacccgtgtctggctggaatactacggac
tcgacatctatccggaacgagcattctgtatttttaccgccaagcgcgaaaattccagtattctccagga
aggcgcactggcagacGTAAGTTGATTCTCCGTCACGCCCACTTTTCTGGCGGGAATTTAAAAAATTTCA
Gatttatactgtggacaatcgactatcggcggcagttggctaccaagatggggatggacgaaaaaattgc
gatccactctgcgacttgaacagcccctttcacttgttagcgGTAGGTGGTGGTCTAGGGTGTCATTTTT
CGATTTTTTCAATTATTCGATGTTTTTAGTGAAAATCGAAAAATCTAAAAATTGAAAATCGAAAAATGAA
AGAAACATTGTTTTTTGGGGACCAAACATCTTAATGAATTTAACAACAGGGAAAACTGAACAGAAACCTG
GACGGTCTTATCCCATTTATCTATATTCTTAAAATGAATGATGGAGAAAAAAGTTAAAATAAAAACATTA
TCAGCTTTTTGTAAGTTTTTCTCAAAAATTGTTCGATTTTTCGATTTTCTAAAAAGTCGAAAAACCGAAA
CCCTTGGTGGTGGTGGTGGTGGACTAGAAAACTCTTCAACGACCACATGGCAATTTTCAGaatttgacgc
ggagaaacaatggtaccacaagtgtattcacctatccggatatgccatatagcggactggatattttcct
gggacttcacttgagtaatgcggattttggtaagattttttttgaaatgttaaatgaaaagttgaaaaat
agtttttatgatttagccactttccagttaaaatttcatttttttaactataaaaagttctggaaaaatg
aatttctAGgccgccgatcctaaaAGTgcaccatttcgcAGaAGTacGTacAGTttcccatctatccctA
GTgGTcttGTtttctgcattctctgcttcacGTgcttGTtttGTtggAGcgcGTttgcatgctgctttaa
attctgaaatattaaaaaaatttcgaAGTttttcAGcacatgggatgggAGTtttgaatttcaatttttt
aaaaacatttttctGTgattAGTgccGTcGTggcacggctGTtAGccgcctatccgGTttattcgatact
ttGTGAGTTTTTTGTAACTTTATGGTCGTCGAAATGGGAAAACTTGGCCACCAATATAAGTTTGGAAAAC
AATTTCCTAAAAATAAAATAATTGAACTTTTCCGATGAATAAAAAAATCGATCAGATATTCTGGAAAAAA
AATCGATAAATTAATCGATTTTCTTGGAAAATACATCGAAAAATTGAGAAAAATAGAAAAATGAATGTTT
TTCGATTACCGATTTATTGATTTTTCGTGAAAACTGAGTTCAGATAATTTTAAAAGCAATGTTTTTCATT
TTTCAAATCAGAATCACTATAGTTTTGAAAAATCAATAATTAATTTATTGATTTTTCAATATAATTTTTT
GGAAAAAATAGAAAAATCCCTTTCTAAAAGTTTTAAATTTCCAAGAAAAATTCATTTTCAAAATCACCAA
CGCGCTCTATAGAGTAGTCGATGAAAATCTCCGTTAAGGGTGCATGGGCAAAACGCGCTCGAACGACAAT
TGTTATTGTATGTTTGGTCTTGCAACGAAAAGTTTGAAAAATTGAAAAAAAGTTGTGTCTGATACATTTT
TTTTTGGCATTTTCTGCTATTTTACACCAGAAAAAATTTAATAAACATAAAAAATCGAAATTTTTCAAGT
TGGACAATTTTCAGtgAGcatcttatccatcctAGTtctcAGTtcAGgacttGTgcacattcGTttAGAG
ccAGatattcgcaaAGccttttcaccggatgattcAGatgctggatAGTAAGTGACTACTGACCTTGAAG
CCTCCTTCCTCCACCAGTCAGAAATAACACGTTTTTTCGCAATGTTTTTCTTTTTCTAATTCGATTTCCC
TTTCTCCCTTTCTTATTGTGATTTGGTCAATGTTTGGTTGACTGGGAAGAAAATTGAATTTTTTTGGAAT
TCCACTTGAAGTTAAAAAACCCAAAATAAATATTTGATCAAAAATAAATAAGAAAAAAAAGAAAACTTTA
AAGCAAATGAAAATTTCGTTCGTAACTATTTTGTTAATTTTTTTAAAACTCCTATTTTAAATATATGCTT
TTTGCGGAAATTTCTATAAATTTTTTTACATTTTTCAGTgaaacccGTGTctggctggaatactacggac
tcgacatctatccggaacgAGcattctGTatttttaccgccaAGcgcgaaaattccAGTattctccAGga
AGgcgcactggcAGacGTAAGTTGATTCTCCGTCACGCCCACTTTTCTGGCGGGAATTTAAAAAATTTCA
GatttatactGTggacaatcgactatcggcggcAGTtggctaccaAGatggggatggacgaaaaaattgc
gatccactctgcgacttgaacAGcccctttcacttGTtAGcgGTAGGTGGTGGTCTAGGGTGTCATTTTT
CGATTTTTTCAATTATTCGATGTTTTTAGTGAAAATCGAAAAATCTAAAAATTGAAAATCGAAAAATGAA
AGAAACATTGTTTTTTGGGGACCAAACATCTTAATGAATTTAACAACAGGGAAAACTGAACAGAAACCTG
GACGGTCTTATCCCATTTATCTATATTCTTAAAATGAATGATGGAGAAAAAAGTTAAAATAAAAACATTA
TCAGCTTTTTGTAAGTTTTTCTCAAAAATTGTTCGATTTTTCGATTTTCTAAAAAGTCGAAAAACCGAAA
CCCTTGGTGGTGGTGGTGGTGGACTAGAAAACTCTTCAACGACCACATGGCAATTTTCAGaatttgacgc
ggAGaaacaatgGTaccacaAGTGTattcacctatccggatatgccatatAGcggactggatattttcct
gggacttcacttgAGTaatgcggattttgGTaAGattttttttgaaatGTtaaatgaaaAGTtgaaaaat
AGTttttatgatttAGccactttccAGTtaaaatttcatttttttaactataaaaAGTtctggaaaaatG
Segnali per il riconoscimento degli introni
Motivi conservati
I segnali dei siti di splicing sono ben
conservati tra le specie
probabilmente la comparsa del
meccanismo di splicing è molto antica
aaggtaaaaa
ttggtaaagc
aacgtaaatt
ctggtaacta
agagtaagac
ccggtaagca
gaagtaagct
aaggtaaggg
atggtaagtc
gcggtaagtt
gaggtaatga
agagtaattg
atggtacata
cgggtacctg
atggtacgga
acagtacgtc
gaggtactct
atggtagaga
ggggtaggaa
atggtaggct
acagtaggtc
caggtagtcc
aaggtatagt
atggtatcct
tgggtatgag
aaagtatgga
caagtatgtc
acggtattaa
aaggtcaaag
acagtcagta
tgggtccgca
agggtctctg
aaggtgaacg
ttggtgagaa
aaggtgagca
tcggtgagct
actgtgaggg
gtagtgagtc
atggtgatga
acggtgccta
cctgtgcgta
caggtgctga
ctggtgggca
ctcgtgggtg
ttggtgtgcc
aaggttaaaa
ggggttagta
aaggttccgt
taggttggag
caggtttcta
Frequency
CAGgtgagtg
5’splice sites
CAGgtaagaa
AAGgtaagaa
CAGgtaagag
CAGgtgaggg
CAGgtgaggc
CAGgtgagcc
aaaaaaagaa
gtaataagga
accccaaggg
cttcgaagga
ttccaaaggc
tatgaaaggt
atctgaagtc
cgtttaagct
gttttaagga
tgctgaagaa
aacaacagtt
caaaacagta
cgtatcagta
gccatcagaa
tacagcagtt
tgtaacagca
aaccgcaggg
atgctcagga
ccccgcaggc
ctacacagct
gcactcaggt
gtacccagtt
tatctcagcc
tgccgcagcc
tttcgcagga
catgacagaa
ctggtcaggc
gtcgacaggt
tgggccaggt
aactgcaggc
atatgcagaa
cattgcaggt
cgctacaggg
ctttccagta
ggattcagga
gttttcaggc
tcctacaggg
tgttgcagcc
caccggagga
aacattagac
ccaactagca
gcaaatagtg
tccactagag
ttgattagag
atccctaggg
ccccctagtc
cttcgtagac
gtgcatagct
tctcatagcg
tttcctaggt
cgggctagca
tctgatagaa
aattttaggc
atctctagag
ccatatagca
ctctgtagac
gcctctagat
gtctatagtg
tcatctagcc
tgttataggt
Frequency
tttttcagGT
3’splice sites
ccctgcagGT
ccccacagGT
ttttttagGT
ctctgcagGT tctttcagAT
ctccccagGC
1
34
67
100
133
166
199
232
265
298
331
364
397
430
463
496
529
562
595
628
661
694
727
760
793
826
859
892
925
958
991
1024
1057
1090
1123
1156
1189
1222
1255
1288
1321
1354
1387
1420
1453
1486
1519
1552
1585
1618
1651
1684
1717
1750
1783
1816
1849
1882
1915
1948
Frequency
exon length (nt)
introns length (nt)
2
7
12
17
22
27
32
37
42
47
52
57
62
67
72
77
82
87
92
97
102
107
112
117
122
127
132
137
142
147
152
157
162
167
172
177
182
187
192
197
202
207
212
217
222
227
232
237
242
247
252
257
262
267
272
277
282
287
292
297
302
307
312
317
322
327
332
337
342
347
Frequency
One point
mutation at
a time
BRCA1 exon 18
100%
20%
17 18 19
80%
17 19
17 18 19
Binding of DAZAP1 and hnRNPA1/A2 to an Exonic Splicing Silencer in a Natural BRCA1 Exon 18 Mutant
Goina E, Skoko N, Pagani F. Mol Cell Biol 2008; 28: 3850–3860
Two point
mutations
at a time
BRCA1 exon 18
Complete exon 18 skipping
Decreased efficiency
Binding of DAZAP1 and hnRNPA1/A2 to an Exonic Splicing Silencer in a Natural BRCA1 Exon 18 Mutant
Goina E, Skoko N, Pagani F. Mol Cell Biol 2008; 28: 3850–3860
Effect of variations in CFTR exon 9
pathological
pathological
100
90
80
70
60
50
40
30
20
10
0
5’-ACAGTTGTTGGCGGTTG-3’
TACCACCC TTATT
GGTTC AA CCGC
G G
T
pathological
% exon 9 inclusion
WT
A G T G A G T C T C G C A C A C A C C T T C A G T T C T
WT 144A 145C
146A
147G
148T 149T150G 151T 153G 154G 155C 156G 157G
ex9 +
ex9 -
Pagani, F., Buratti, E., Stuani, C., and Baralle, F. E. (2003) J Biol Chem
Pagani, F., Stuani, C., Zuccato, E., Kornblihtt, A. R., and Baralle, F. E. (2003) J Biol Chem
An additional exonic constraints: the splicing code
The genetic code is degenerate, but it is not all rodustness
. . . Ala Val
Arg . . .
GCA GTA CGA
C
C
C
G
G
G
T
T
T
AGA
G
4 * 4 * 6 = 96
Three AAs specified by 96
synonymous words
GCAGTACGA
GCAGTACGC
GCAGTACGG
GCAGTACGT
GCAGTAAGA
GCAGTAAGG
GCAGTCCGA
GCAGTCCGC
GCAGTCCGG
GCAGTCCGT
GCAGTCAGA
GCAGTCAGG
GCAGTGCGA
GCAGTGCGC
GCAGTGCGG
GCAGTGCGT
GCAGTGAGA
GCAGTGAGG
GCAGTTCGA
GCAGTTCGC
GCAGTTCGG
GCAGTTCGT
GCAGTTAGA
GCAGTTAGG
GCCGTACGA
GCCGTACGC
GCCGTACGG
GCCGTACGT
GCCGTAAGA
GCCGTAAGG
GCCGTCCGA
GCCGTCCGC
GCCGTCCGG
GCCGTCCGT
GCCGTCAGA
GCCGTCAGG
GCCGTGCGA
GCCGTGCGC
GCCGTGCGG
GCCGTGCGT
GCCGTGAGA
GCCGTGAGG
GCCGTTCGA
GCCGTTCGC
GCCGTTCGG
GCCGTTCGT
GCCGTTAGA
GCCGTTAGG
GCGGTACGA
GCGGTACGC
GCGGTACGG
GCGGTACGT
GCGGTAAGA
GCGGTAAGG
GCGGTCCGA
GCGGTCCGC
GCGGTCCGG
GCGGTCCGT
GCGGTCAGA
GCGGTCAGG
GCGGTGCGA
GCGGTGCGC
GCGGTGCGG
GCGGTGCGT
GCGGTGAGA
GCGGTGAGG
GCGGTTCGA
GCGGTTCGC
GCGGTTCGG
GCGGTTCGT
GCGGTTAGA
GCGGTTAGG
GCTGTACGA
GCTGTACGC
GCTGTACGG
GCTGTACGT
GCTGTAAGA
GCTGTAAGG
GCTGTCCGA
GCTGTCCGC
GCTGTCCGG
GCTGTCCGT
GCTGTCAGA
GCTGTCAGG
GCTGTGCGA
GCTGTGCGC
GCTGTGCGG
GCTGTGCGT
GCTGTGAGA
GCTGTGAGG
GCTGTTCGA
GCTGTTCGC
GCTGTTCGG
GCTGTTCGT
GCTGTTAGA
GCTGTTAGG
cryptic exon
exon31
NF1
gene
ttttatagTGAGAATA
A>G
WT MUT
La mutazione
attiva un esone
criptico (in
rosso)
Raponi M, Upadhyaya M, Baralle D.
Functional splicing assay shows a pathogenic intronic mutation in neurofibromatosis type 1 (NF1) due to intronic sequence exonization.
Hum Mutat. 2006; 27(3):294-295.
cryptic exon
exon31
NF1
gene
Disruption
of 5’ss
restores
normal
splicing
TAGataata
TAGgtggga
TAGgtaata
CAGgtattg
CAAgtattg
CAAgtaagc
CAAgtaagg
La seq 2 ha un sito di
splicing in 5’ più debole
della seq 1. La seq 3
non ha il sito.
Raponi M, Upadhyaya M, Baralle D.
Functional splicing assay shows a pathogenic intronic mutation in neurofibromatosis type 1 (NF1) due to intronic sequence exonization.
Hum Mutat. 2006;27(3):294-295.
ATM gene
structure
20
WT: GGCCAGGTAAGTGATA
20
mutations
21
21
DEL: GGCCAG____GTGATA
MUT: GGCCAGGTCTGTGATA
M WT del mut
results
A new type of mutation causes a
splicing defect in ATM
Pagani F, Buratti E, Stuani C, Bendix R,
Dörk T, Baralle FE
Nature Genetics 2002, 30: 426-429
20
20 21
21
Many elements regulate the splicing process
exonic splicing enhancer ESE
exonic splicing silencer ESS
intronic splicing enhancer ISE
intronic splicing silencer ISS
A compact formalism, but…
score
matrix
Compression and reconstruction of motifs
Experimental
assessed
binding
sites
AGG
AGT
CGT
AGG
CGT
zip
consensus
sequence
A G
G
C T
unzip
AGG
AGT
CGG
CGT
Intron definition / exon definition
Modello di exonic splicing enhancer mediato da proteine SR
Modello di exonic splicing silencer
elements promoting exons
elements promoting introns
ESE, ISS: esone
ESS, ISE: introne
PROTEINS REGULATING SPLICING STORED IN SPLICEAID
9G8, CUG-BP1, DAZAP1, ETR-3, Fox-1, Fox-2,
FMRP, hnRNP A0, hnRNP A1, hnRNP A2/B1, hnRNP
C, hnRNP C1, hnRNP C2, hnRNP D, hnRNP D0,
hnRNP DL, hnRNP E1, hnRNP E2, hnRNP F, hnRNP
G, hnRNP H1, hnRNP H2, hnRNP I (PTB), hnRNP J,
hnRNP K, hnRNP L, hnRNP LL, hnRNP M, hnRNP P
(TLS), hnRNP Q, hnRNP U, HTra2alpha, HTra2beta1,
HuB, HuD, HuR, KSRP, MBNL1, Nova-1, Nova-2,
nPTB, PSF, RBM4, RBM25, Sam68, SAP155, SC35,
SF1, SF2/ASF, SLM-1, SLM-2, SRp20, SRp30c,
SRp38, SRp40, SRp54, SRp55, SRp75, TDP43, TIA-1,
TIAL1, YB-1, ZRANB2 …
Some comparisons among literature data (SpliceAid) and prediction tools
SEQUENCE
ACAAC
SPLICEAID
EXPERIMENTALLY
ASSESSED BINDING
YB-1
COMPETING TOOLS
Splicing
ESE Finder Rescue ESE
Rainbow
no binding no ESE
SRp40
GAAGAAGA
HTra2A, HTra2B1,
SF2/ASF, SC35,
no binding
SRp40, SRp55, SRp75
CUGGCGUCGUCGC
no binding
UGACUG
hnRNP A1
3 ESE
Tra2B
SF2/ASF,
SRp55
2 ESE
SRp40, SRp55
no binding
no ESE
SRp40, SRp55
UUUUAGACAA
hnRNP C1, Sam68,
hnRNP A1, hnRNP D,
no binding
hnRNP E1, hnRNP E2,
SRp38
1 ESE
hnRNP A2/B1,
hnRNP C1/C2,
hnRNP E1/E2,
SRp40, SRp55,
U2AF65
UGUGUGUGUGUGUGUGUG
CUG-BP1, ETR-3,
TDP43
no ESE
hnRNP U
SRp55
Pan troglodytes
average nucleotide divergence of just 1.2%
Suggested papers
Nature reviews. Genetics. 2002; 3(4): 285-298
Listening to silence and understanding nonsense: exonic
mutations that affect splicing.
Cartegni L, Chew SL, Krainer AR.
PMID: 11967553
Nature reviews. Genetics. 2007; 8(10): 749-761.
Splicing in disease: disruption of the splicing code and the
decoding machinery.
Wang GS, Cooper TA.
PMID: 17726481