Università
Politecnica
delle Marche
Istituto di Biologia e
Genetica
Lo splicing dell’RNA
• definizione
• importanza
• predizione
Francesco Piva
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
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
basi
gtttttt
gtttctg
gttcgtt
gttgttc
gtgagaa
gttgcta
gtgtgtt
gttctgt
gtgggtt
gttccgg
gttatgc
gttacga
gtgttct
gtgcgtt
gtgtccg
gtggtcc
gtggcca
gtcagtt
gtgctat
gtgccag
gtgatac
0,10
gtgacaa
gtaggtt
gtctgtt
gtctatg
gtcggtc
gtcgata
gtccggt
gtccagg
gtatttt
gtcaggc
gtcaaga
gtacgtt
gtatgct
gtatacg
gtaattt
gtaggcc
gtagaca
gtacgat
gtacaag
0,02
gtaagac
gtaaaaa
frequenze
Sequenze in testa negli introni di C.elegans
gtaagtt
gtgagtt
0,08
0,06
0,04
gttagtt
gtatgtt
gtttgtt
gttggtt
0,00
basi
ttgaaag
ttaaaag
agttcag
tggaaag
tattcag
tgaaaag
tcgaaag
tcaaaag
aattcag
tagaaag
taaaaag
ctttcag
gtgaaag
gtaaaag
gggaaag
ggaaaag
gcgaaag
gcaaaag
gagaaag
gaaaaag
0,05
ctgaaag
ctaaaag
cggaaag
cgaaaag
ccgaaag
ccaaaag
cagaaag
caaaaag
atgaaag
ataaaag
acttcag
aggaaag
agaaaag
acgaaag
acaaaag
aagaaag
aaaaaag
frequenze
Sequenze in coda negli introni di C.elegans
ttttcag
0,25
0,20
0,15
atttcag
0,10
tttccag
gtttcag
tcttcag
0,00
Terne che precedono gli introni in C.elegans
0,14
0,12
0,08
0,06
0,04
0,02
basi
ttt
att
cgt
gct
tat
aat
ctg
ggg
tcg
acg
cag
gtc
tgc
agc
ccc
gac
tta
ata
cga
gca
taa
0,00
aaa
frequenze
0,10
Distribuzione dimensioni introni C.elegans
0,09
0,08
0,07
frequenze
0,06
0,05
0,04
0,03
0,02
0,01
0,00
222233344455556667778888999111111111111111111111111
036925814703692581470369258000111122233344445556667
147036925814703692581470
dimensioni
Meccanismo
dello splicing
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
Alcuni modi di fare
splicing alternativo
Alcuni genomi virali
subiscono splicing
all’interno della cellula
ospite
equine infectious anemia virus (EIAV)
Effect of synonymous variations at CERES in CFTR exon 9
% exon 9 inclusion
WT
100
90
80
70
60
50
40
30
20
10
0
5’-ACAGTTGTTGGCGGTTG-3’
TACCACCC TTATT
point mutations
GGTTC AA CCGC
G G
T
Q452P
V456E F
A455E
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 278, 1511
The majority of random substitutions at the synonymous codons in
CFTR exon 12 induce exon inclusion/1
WT
AAA GAT GCT GAT TTG TAT TTA TTA GAC TCT CCT TTT GGA
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
AAG
AAG
AAA
AAG
AAG
AAG
AAA
AAA
AAA
AAG
AAA
AAA
GAC
GAC
GAC
GAC
GAC
GAC
GAC
GAC
GAT
GAC
GAC
GAC
GCA
GCG
GCT
GCG
GCC
GCC
GCG
GCA
GCC
GCT
GCA
GCA
GAT
GAC
GAT
GAC
GAC
GAC
GAT
GAT
GAC
GAC
GAC
GAC
TTG
TTG
TTG
TTG
TTG
TTA
TTG
TTA
TTA
TTG
TTG
TTA
TAC
TAT
TAC
TAC
TAT
TAC
TAT
TAT
TAT
TAT
TAT
TAC
TTA
TTA
TTA
TTA
TTG
TTG
TTA
TTG
TTG
TTA
TTG
TTA
TTA
TTA
TTG
TTG
TTG
TTG
TTG
TTG
TTG
TTG
TTG
TTG
GAT
GAT
GAT
GAC
GAC
GAC
GAT
GAC
GAT
GAC
GAC
GAC
TCA
TCG
TCA
TCC
TCT
TCG
TCA
TCC
TCA
TCC
TCA
TCA
CCC
CCG
CCG
CCC
CCG
CCT
CCT
CCG
CCC
CCA
CCG
CCG
TTC
TTC
TTC
TTC
TTC
TTT
TTC
TTT
TTC
TTT
TTC
TTT
GGA
GGC
GGA
GGT
GGT
GGC
GGC
GGA
GGC
GGG
GGT
GGT
% exon inclusion
100%
80%
60%
40%
20%
0%
GAT
GAC
GAT
GAT
GAT
TTG
TTG
TTA
TTG
TTA
TAT
TAT
TAT
TAT
TAC
TTA
TTA
TTA
TTG
TTG
TTG
TTG
TTA
TTA
TTG
GAT
GAT
GAC
GAC
GAT
TCT
TCG
TCT
TCA
TCC
CCG
CCA
CCG
CCG
CCC
TTT
TTT
TTC
TTT
TTC
GGG
GGT
GGT
GGC
GGC
A1 A3 A5 A7 A9 A11 A13 A15 A17 A19 A21
A1->12
WT
GCA
GCG
GCT
GCG
GCA
A21
GAC
GAT
GAT
GAT
GAC
A18->20
AAA
AAA
AAG
AAA
AAA
A13->17
A13
A14
A15
A16
A17
A18 AAA GAT GCA GAT TTG TAC TTG TTA GAC TCG CCC TTT GGC
A19 AAG GAC GCA GAT TTG TAT TTG TTA GAC TCC CCA TTC GGG
A20 AAG GAC GCT GAC TTA TAC TTG TTA GAT TCC CCT TTC GGT
A21 AAG GAT GCA GAT TTA TAT TTA TTA GAC TCC CCT TTT GGT
• Changes in splicing efficiency are not related to the use of unpreferred synonymous codons (underlined)
• Nucleotide changes have different effect according to the context in which they occur
Intron definition / exon definition
Modello di exonic splicing enhancer mediato da proteine SR
Modello di exonic splicing silencer
Ricombinazione e splicing alternativo
In presenza di una struttura interrotta, mutazioni neutre possono originare nuove forme di alternative splicing senza distruggere le
forme funzionali già esistenti. Questa rappresenta un’opportunità per l’evoluzione di esplorare nuovi schemi aggiungendoli
eventualmente a quelli precedenti.
(Pagani F, Raponi M, Baralle FE. Synonymous mutations in CFTR exon 12 affect splicing and are not neutral in evolution Proc
Natl Acad Sci U S A. 2005 May 3;102(18):6368-72.)
Molte altre proteine partecipano allo splicing
9G8, CUG-BP1, DAZAP1, ETR-3, Fox-1, Fox-2,
hnRNP-A0, hnRNP-A1, hnRNP-A2/B1, hnRNPC, hnRNP-D, hnRNP-D0, hnRNP DL, hnRNP E1,
hnRNP E2, hnRNP-F, hnRNP G, hnRNP-H1,
hnRNP-H2, hnRNP-I, hnRNP J, hnRNP K,
hnRNP-L, hnRNP M, hnRNP P (TLS), hnRNP Q,
hnRNP U, HTra2beta1, HuB, HuD, HuR, KSRP,
Nova-1, Nova-2, nPTB, PSF, Sam68, SC35, SF1,
SF2/ASF, SLM-1, SLM-2, SRp20, SRp30c,
SRp38, SRp40, SRp54, SRp55, SRp75, TDP43,
TIA-1, TIAL1, YB-1 …
ESE, ISS: esone
ESS, ISE: introne
Pan troglodytes
average nucleotide divergence of just 1.2%
Letture consigliate
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