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