Post on 14-Jan-2016
description
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 esone2 introne2 esone3
esone1
esone1
introne1
esone2
esone2
introne2
esone3
esone3
SPLICING
eliminazione introni
unione esoni
GT GTAG AG
Lo splicing avviene in tutto il trascritto, anche nelle zone non codificanti
attggaaaccgaaacccgttggtcacctctgcaatagccctccctccctcacttctacaattttgtgacagtggtcttgttttctgcattctctgcttcacgtgcttgttttgttggagcgcgtttgcatgctgctttaaattctgaaatattaaaaaaatttcgaagtttttcagcacatgggatgggagttttgaatttcaattttttaaaaacatttttctgtgattagtgccgtcgtggcacggctgttagccgcctatccggtttattcgatactttGTGAGTTTTTTGTAACTTTATGGTCGTCGAAATGGGAAAACTTGGCCACCAATATAAGTTTGGAAAACAATTTCCTAAAAATAAAATAATTGAACTTTTCCGATGAATAAAAAAATCGATCAGATATTCTGGAAAAAAAATCGATAAATTAATCGATTTTCTTGGAAAATACATCGAAAAATTGAGAAAAATAGAAAAATGAATGTTTTTCGATTACCGATTTATTGATTTTTCGTGAAAACTGAGTTCAGATAATTTTAAAAGCAATGTTTTTCATTTTTCAAATCAGAATCACTATAGTTTTGAAAAATCAATAATTAATTTATTGATTTTTCAATATAATTTTTTGGAAAAAATAGAAAAATCCCTTTCTAAAAGTTTTAAATTTCCAAGAAAAATTCATTTTCAAAATCACCAACGCGCTCTATAGAGTAGTCGATGAAAATCTCCGTTAAGGGTGCATGGGCAAAACGCGCTCGAACGACAATTGTTATTGTATGTTTGGTCTTGCAACGAAAAGTTTGAAAAATTGAAAAAAAGTTGTGTCTGATACATTTTTTTTTGGCATTTTCTGCTATTTTACACCAGAAAAAATTTAATAAACATAAAAAATCGAAATTTTTCAAGTTGGACAATTTTCAGtgagcatcttatccatcctagttctcagttcaggacttgtgcacattcgtttagagccagatattcgcaaagccttttcaccggatgattcagatgctggataGTAAGTGACTACTGACCTTGAAGCCTCCTTCCTCCACCAGTCAGAAATAACACGTTTTTTCGCAATGTTTTTCTTTTTCTAATTCGATTTCCCTTTCTCCCTTTCTTATTGTGATTTGGTCAATGTTTGGTTGACTGGGAAGAAAATTGAATTTTTTTGGAATTCCACTTGAAGTTAAAAAACCCAAAATAAATATTTGATCAAAAATAAATAAGAAAAAAAAGAAAACTTTAAAGCAAATGAAAATTTCGTTCGTAACTATTTTGTTAATTTTTTTAAAACTCCTATTTTAAATATATGCTTTTTGCGGAAATTTCTATAAATTTTTTTACATTTTTCAGtgaaacccgtgtctggctggaatactacggactcgacatctatccggaacgagcattctgtatttttaccgccaagcgcgaaaattccagtattctccaggaaggcgcactggcagacGTAAGTTGATTCTCCGTCACGCCCACTTTTCTGGCGGGAATTTAAAAAATTTCAGatttatactgtggacaatcgactatcggcggcagttggctaccaagatggggatggacgaaaaaattgcgatccactctgcgacttgaacagcccctttcacttgttagcgGTAGGTGGTGGTCTAGGGTGTCATTTTTCGATTTTTTCAATTATTCGATGTTTTTAGTGAAAATCGAAAAATCTAAAAATTGAAAATCGAAAAATGAAAGAAACATTGTTTTTTGGGGACCAAACATCTTAATGAATTTAACAACAGGGAAAACTGAACAGAAACCTGGACGGTCTTATCCCATTTATCTATATTCTTAAAATGAATGATGGAGAAAAAAGTTAAAATAAAAACATTATCAGCTTTTTGTAAGTTTTTCTCAAAAATTGTTCGATTTTTCGATTTTCTAAAAAGTCGAAAAACCGAAACCCTTGGTGGTGGTGGTGGTGGACTAGAAAACTCTTCAACGACCACATGGCAATTTTCAGaatttgacgcggagaaacaatggtaccacaagtgtattcacctatccggatatgccatatagcggactggatattttcctgggacttcacttgagtaatgcggattttggtaagattttttttgaaatgttaaatgaaaagttgaaaaatagtttttatgatttagccactttccagttaaaatttcatttttttaactataaaaagttctggaaaaatg
aatttctAGgccgccgatcctaaaAGTgcaccatttcgcAGaAGTacGTacAGTttcccatctatccctAGTgGTcttGTtttctgcattctctgcttcacGTgcttGTtttGTtggAGcgcGTttgcatgctgctttaaattctgaaatattaaaaaaatttcgaAGTttttcAGcacatgggatgggAGTtttgaatttcaattttttaaaaacatttttctGTgattAGTgccGTcGTggcacggctGTtAGccgcctatccgGTttattcgatactttGTGAGTTTTTTGTAACTTTATGGTCGTCGAAATGGGAAAACTTGGCCACCAATATAAGTTTGGAAAACAATTTCCTAAAAATAAAATAATTGAACTTTTCCGATGAATAAAAAAATCGATCAGATATTCTGGAAAAAAAATCGATAAATTAATCGATTTTCTTGGAAAATACATCGAAAAATTGAGAAAAATAGAAAAATGAATGTTTTTCGATTACCGATTTATTGATTTTTCGTGAAAACTGAGTTCAGATAATTTTAAAAGCAATGTTTTTCATTTTTCAAATCAGAATCACTATAGTTTTGAAAAATCAATAATTAATTTATTGATTTTTCAATATAATTTTTTGGAAAAAATAGAAAAATCCCTTTCTAAAAGTTTTAAATTTCCAAGAAAAATTCATTTTCAAAATCACCAACGCGCTCTATAGAGTAGTCGATGAAAATCTCCGTTAAGGGTGCATGGGCAAAACGCGCTCGAACGACAATTGTTATTGTATGTTTGGTCTTGCAACGAAAAGTTTGAAAAATTGAAAAAAAGTTGTGTCTGATACATTTTTTTTTGGCATTTTCTGCTATTTTACACCAGAAAAAATTTAATAAACATAAAAAATCGAAATTTTTCAAGTTGGACAATTTTCAGtgAGcatcttatccatcctAGTtctcAGTtcAGgacttGTgcacattcGTttAGAGccAGatattcgcaaAGccttttcaccggatgattcAGatgctggatAGTAAGTGACTACTGACCTTGAAGCCTCCTTCCTCCACCAGTCAGAAATAACACGTTTTTTCGCAATGTTTTTCTTTTTCTAATTCGATTTCCCTTTCTCCCTTTCTTATTGTGATTTGGTCAATGTTTGGTTGACTGGGAAGAAAATTGAATTTTTTTGGAATTCCACTTGAAGTTAAAAAACCCAAAATAAATATTTGATCAAAAATAAATAAGAAAAAAAAGAAAACTTTAAAGCAAATGAAAATTTCGTTCGTAACTATTTTGTTAATTTTTTTAAAACTCCTATTTTAAATATATGCTTTTTGCGGAAATTTCTATAAATTTTTTTACATTTTTCAGTgaaacccGTGTctggctggaatactacggactcgacatctatccggaacgAGcattctGTatttttaccgccaAGcgcgaaaattccAGTattctccAGgaAGgcgcactggcAGacGTAAGTTGATTCTCCGTCACGCCCACTTTTCTGGCGGGAATTTAAAAAATTTCAGatttatactGTggacaatcgactatcggcggcAGTtggctaccaAGatggggatggacgaaaaaattgcgatccactctgcgacttgaacAGcccctttcacttGTtAGcgGTAGGTGGTGGTCTAGGGTGTCATTTTTCGATTTTTTCAATTATTCGATGTTTTTAGTGAAAATCGAAAAATCTAAAAATTGAAAATCGAAAAATGAAAGAAACATTGTTTTTTGGGGACCAAACATCTTAATGAATTTAACAACAGGGAAAACTGAACAGAAACCTGGACGGTCTTATCCCATTTATCTATATTCTTAAAATGAATGATGGAGAAAAAAGTTAAAATAAAAACATTATCAGCTTTTTGTAAGTTTTTCTCAAAAATTGTTCGATTTTTCGATTTTCTAAAAAGTCGAAAAACCGAAACCCTTGGTGGTGGTGGTGGTGGACTAGAAAACTCTTCAACGACCACATGGCAATTTTCAGaatttgacgcggAGaaacaatgGTaccacaAGTGTattcacctatccggatatgccatatAGcggactggatattttcctgggacttcacttgAGTaatgcggattttgGTaAGattttttttgaaatGTtaaatgaaaAGTtgaaaaatAGTttttatgatttAGccactttccAGTtaaaatttcatttttttaactataaaaAGTtctggaaaaatG
Segnali per il riconoscimento degli introni
Motivi conservati
I segnali dei siti di splicing sono ben conservati tra le specieprobabilmente la comparsa del meccanismo di splicing è molto antica
Sequenze in testa negli introni di C.elegans
gtaagtt
gtaattt
gtacgtt
gtaggtt
gtatgtt
gtattttgtcagtt
gtgagtt
gtgggtt
gttagtt
gtgagaa
gttcgttgttggtt
gtttgtt
gtgtgttgtgcgtt
0,00
0,02
0,04
0,06
0,08
0,10
gtaaaaa
gtaagac
gtacaag
gtacgat
gtagaca
gtaggcc
gtatacg
gtatgct
gtcaaga
gtcaggc
gtccagg
gtccggt
gtcgata
gtcggtc
gtctatg
gtctgtt
gtgacaa
gtgatac
gtgccag
gtgctat
gtggcca
gtggtcc
gtgtccg
gtgttct
gttacga
gttatgc
gttccgg
gttctgt
gttgcta
gttgttc
gtttctg
gtttttt
basi
frequ
enze
Sequenze in coda negli introni di C.elegans
tttccag
ttttcag
tcttcagtattcag
gtttcagctttcag
atttcag
agttcagacttcagaattcag
0,00
0,05
0,10
0,15
0,20
0,25
aaaaaag
aagaaag
acaaaag
acgaaag
agaaaag
aggaaag
ataaaag
atgaaag
caaaaag
cagaaag
ccaaaag
ccgaaag
cgaaaag
cggaaag
ctaaaag
ctgaaag
gaaaaag
gagaaag
gcaaaag
gcgaaag
ggaaaag
gggaaag
gtaaaag
gtgaaag
taaaaag
tagaaag
tcaaaag
tcgaaag
tgaaaag
tggaaag
ttaaaag
ttgaaag
basi
freq
uenz
e
Terne che precedono gli introni in C.elegans
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
aaa
taa
gca
cga
ata
tta
gac
ccc
agc
tgc
gtc
cag
acg
tcg
ggg
ctg
aat
tat
gct
cgt
att
ttt
basi
freq
uenz
e
Distribuzione dimensioni introni C.elegans
0,00
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,08
0,09
202326293235384144475053565962656871747780838689929598101
104
107
110
113
116
119
122
125
128
131
134
137
140
143
146
149
152
155
158
161
164
167
170
dimensioni
freq
uenz
e
Meccanismo dello splicing
U2AF
U2AF
U2AFU2AF
arly
U2AF si lega al tratto pirimidinico a valle del sito di ramificazione
snRNP U2 si lega al sito di ramificazione (richiesta idrolisi ATP)
Arg-Ser
le prot SR connettono U2Af con snRNP U1
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
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
il 3’ss è tagliato e gli esoni vengono saldati insieme, il cappio verrà deramificato
introne (5’ss)
Sm protein
snRNP U1
C5
G16
RBD: RNA binding domain
si appaia al sito di ramificazione
Sm protein
snRNP U2
si appaiano con snRNA U6
U5
U17
3 541 2
1 2 3 5Muscolo cardiaco
1 43 5Muscolo uterino
Lo splicing è tessuto specifico
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)
WT 5’-ACAGTTGTTGGCGGTTG-3’TACCACCC TTATTGGTTC AA CCGC G G T
point mutations
0102030405060708090
100
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 144A145C 146A 147G 148T 149T150G 151T 153G 154G 155C 156G 157G
ex9+
ex9-
% e
xon
9 in
clu
sion
A455E
V456EF
Q452P
Effect of synonymous variations at CERES in CFTR exon 9
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
A
0%
20%
40%
60%
80%
100%
A1 A3 A5 A7 A9 A11 A13 A15 A17 A19 A21
% exon inclusion
A1->12
A13
->17
A18
->20
A21
WT
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 AAG GAC GCA GAT TTG TAC TTA TTA GAT TCA CCC TTC GGAA2 AAG GAC GCG GAC TTG TAT TTA TTA GAT TCG CCG TTC GGCA3 AAA GAC GCT GAT TTG TAC TTA TTG GAT TCA CCG TTC GGAA4 AAG GAC GCG GAC TTG TAC TTA TTG GAC TCC CCC TTC GGTA5 AAG GAC GCC GAC TTG TAT TTG TTG GAC TCT CCG TTC GGTA6 AAG GAC GCC GAC TTA TAC TTG TTG GAC TCG CCT TTT GGCA7 AAA GAC GCG GAT TTG TAT TTA TTG GAT TCA CCT TTC GGCA8 AAA GAC GCA GAT TTA TAT TTG TTG GAC TCC CCG TTT GGAA9 AAA GAT GCC GAC TTA TAT TTG TTG GAT TCA CCC TTC GGCA10 AAG GAC GCT GAC TTG TAT TTA TTG GAC TCC CCA TTT GGGA11 AAA GAC GCA GAC TTG TAT TTG TTG GAC TCA CCG TTC GGTA12 AAA GAC GCA GAC TTA TAC TTA TTG GAC TCA CCG TTT GGT
A13 AAA GAC GCA GAT TTG TAT TTA TTG GAT TCT CCG TTT GGG A14 AAA GAT GCG GAC TTG TAT TTA TTG GAT TCG CCA TTT GGTA15 AAG GAT GCT GAT TTA TAT TTA TTA GAC TCT CCG TTC GGTA16 AAA GAT GCG GAT TTG TAT TTG TTA GAC TCA CCG TTT GGCA17 AAA GAC GCA GAT TTA TAC TTG TTG GAT TCC CCC TTC GGC
A18 AAA GAT GCA GAT TTG TAC TTG TTA GAC TCG CCC TTT GGCA19 AAG GAC GCA GAT TTG TAT TTG TTA GAC TCC CCA TTC GGGA20 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.)
9G8, CUG-BP1, DAZAP1, ETR-3, Fox-1, Fox-2, hnRNP-A0, hnRNP-A1, hnRNP-A2/B1, hnRNP-C, 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 …
Molte altre proteine partecipano allo splicing
ESE, ISS: esone
ESS, ISE: introne
Pan troglodytesaverage nucleotide divergence of just 1.2%
Letture consigliate
Nature reviews. Genetics. 2002; 3(4): 285-298Listening 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