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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
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These are synonymous words, are we sure ?
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 . . . Ala Val Arg . . . GCA C G T GTA C G T CGA C G T AGA 4 * 4 * 6 = 96 Three AAs specified by 96 synonymous words
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Genomic DNA mRNA ATGTAAACGTATATCGTGACAGTGGTCTGTTAGTATTCCTTTAGTCATGGTTT
ATGTAAACTGGTCTGTTATCATGGTTT mRNA
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attggaaaccgaaacccgttggtcacctctgcaatagccctccctccctcacttctacaattttgtgacagtggtcttgttttctgcattctctgcttcacgtgcttgttttgttggagcgcgtttgcatgctgctttaaattctgaaatattaaaaaaatttcgaagtttttcagcacatgggatgggagttttgaatttcaattttttaaaaacatttttctgtgattagtgccgtcgtggcacggctgttagccgcctatccggtttattcgatactttGTGAGTTTTTTGTAACTTTATGGTCGTCGAAATGGGAAAACTTGGCCACCAATATAAGTTTGGAAAACAATTTCCTAAAAATAAAATAATTGAACTTTTCCGATGAATAAAAAAATCGATCAGATATTCTGGAAAAAAAATCGATAAATTAATCGATTTTCTTGGAAAATACATCGAAAAATTGAGAAAAATAGAAAAATGAATGTTTTTCGATTACCGATTTATTGATTTTTCGTGAAAACTGAGTTCAGATAATTTTAAAAGCAATGTTTTTCATTTTTCAAATCAGAATCACTATAGTTTTGAAAAATCAATAATTAATTTATTGATTTTTCAATATAATTTTTTGGAAAAAATAGAAAAATCCCTTTCTAAAAGTTTTAAATTTCCAAGAAAAATTCATTTTCAAAATCACCAACGCGCTCTATAGAGTAGTCGATGAAAATCTCCGTTAAGGGTGCATGGGCAAAACGCGCTCGAACGACAATTGTTATTGTATGTTTGGTCTTGCAACGAAAAGTTTGAAAAATTGAAAAAAAGTTGTGTCTGATACATTTTTTTTTGGCATTTTCTGCTATTTTACACCAGAAAAAATTTAATAAACATAAAAAATCGAAATTTTTCAAGTTGGACAATTTTCAGtgagcatcttatccatcctagttctcagttcaggacttgtgcacattcgtttagagccagatattcgcaaagccttttcaccggatgattcagatgctggataGTAAGTGACTACTGACCTTGAAGCCTCCTTCCTCCACCAGTCAGAAATAACACGTTTTTTCGCAATGTTTTTCTTTTTCTAATTCGATTTCCCTTTCTCCCTTTCTTATTGTGATTTGGTCAATGTTTGGTTGACTGGGAAGAAAATTGAATTTTTTTGGAATTCCACTTGAAGTTAAAAAACCCAAAATAAATATTTGATCAAAAATAAATAAGAAAAAAAAGAAAACTTTAAAGCAAATGAAAATTTCGTTCGTAACTATTTTGTTAATTTTTTTAAAACTCCTATTTTAAATATATGCTTTTTGCGGAAATTTCTATAAATTTTTTTACATTTTTCAGtgaaacccgtgtctggctggaatactacggactcgacatctatccggaacgagcattctgtatttttaccgccaagcgcgaaaattccagtattctccaggaaggcgcactggcagacGTAAGTTGATTCTCCGTCACGCCCACTTTTCTGGCGGGAATTTAAAAAATTTCAGatttatactgtggacaatcgactatcggcggcagttggctaccaagatggggatggacgaaaaaattgcgatccactctgcgacttgaacagcccctttcacttgttagcgGTAGGTGGTGGTCTAGGGTGTCATTTTTCGATTTTTTCAATTATTCGATGTTTTTAGTGAAAATCGAAAAATCTAAAAATTGAAAATCGAAAAATGAAAGAAACATTGTTTTTTGGGGACCAAACATCTTAATGAATTTAACAACAGGGAAAACTGAACAGAAACCTGGACGGTCTTATCCCATTTATCTATATTCTTAAAATGAATGATGGAGAAAAAAGTTAAAATAAAAACATTATCAGCTTTTTGTAAGTTTTTCTCAAAAATTGTTCGATTTTTCGATTTTCTAAAAAGTCGAAAAACCGAAACCCTTGGTGGTGGTGGTGGTGGACTAGAAAACTCTTCAACGACCACATGGCAATTTTCAGaatttgacgcggagaaacaatggtaccacaagtgtattcacctatccggatatgccatatagcggactggatattttcctgggacttcacttgagtaatgcggattttggtaagattttttttgaaatgttaaatgaaaagttgaaaaatagtttttatgatttagccactttccagttaaaatttcatttttttaactataaaaagttctggaaaaatg
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Struttura tipica dei geni umani
esoni introni
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SPLICING eliminazione introni unione esoni esone1 introne1 esone2
GT AG GT AG SPLICING eliminazione introni introne1 introne2 esone2 esone3 esone1 unione esoni esone1 esone2 esone3
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Lo splicing avviene in tutto il trascritto, anche nelle zone non codificanti
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R = G, A Y = T, C
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= + + Meccanismo di splicing O R C ORI HO RII O R C ORII HO RI estere
alcool O + R C ORI HO RII = O + R C ORII HO RI due legami fosfoesterici
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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 si legano insieme U2AF U2AF snRNP U5 si lega al 5’ss, snRNP U6 si lega a snRNP U2 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 snRNP U1 è rilasciato, snRNP U5 si sposta dall’esone all’introne, snRNP U6 si lega al 5’ss
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introne (5’ss)
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snRNP U1 Sm protein
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RBD: RNA binding domain
C5
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snRNP U2 Sm protein si appaiano con snRNA U6
si appaia al sito di ramificazione
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U17 U5
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Lo splicing è tessuto specifico
1 2 3 5 Muscolo cardiaco 1 2 3 4 5 1 4 3 5 Muscolo uterino
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Esempio di alternative splicing di un gene umano
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Alternative splicing tessuto specifico
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Tutti i modi di fare splicing alternativo
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Alcuni genomi virali subiscono splicing all’interno della cellula ospite
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equine infectious anemia virus (EIAV)
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AIM: mRNA structure pre mRNA sequence SPLICING PREDICTION TOOL
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Segnali per il riconoscimento degli introni
Motivi conservati
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I segnali dei siti di splicing sono ben conservati tra le specie
probabilmente la comparsa del meccanismo di splicing è molto antica
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5’splice sites
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One point mutation at a time
BRCA1 exon 18 100% 20% 17 18 19 17 18 19 80% 17 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
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Two point mutations at a time
BRCA1 exon 18 Decreased efficiency Complete exon 18 skipping 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
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Effect of variations in CFTR exon 9
WT 5’-ACAGTTGTTGGCGGTTG-3’ TACCACCC TTATT GGTTC AA CCGC G G T 100 pathological 90 80 pathological 70 % exon 9 inclusion 60 pathological 50 40 30 20 10 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 149T 150G 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
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An additional exonic constraints: the splicing code
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The genetic code is degenerate, but it is not all rodustness
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 . . . Ala Val Arg . . . GCA C G T GTA C G T CGA C G T AGA 4 * 4 * 6 = 96 Three AAs specified by 96 synonymous words
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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):
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cryptic exon exon31 NF1 gene CAGgtattg TAGataata CAAgtattg TAGgtggga
Disruption of 5’ss restores normal splicing TAGataata CAAgtattg TAGgtggga CAAgtaagc TAGgtaata 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):
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ATM gene structure mutations results M WT del mut 20 21
WT: GGCCAGGTAAGTGATA 20 21 mutations DEL: GGCCAG____GTGATA MUT: GGCCAGGTCTGTGATA M WT del mut results 20 21 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: 20 21
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Many elements regulate the splicing process
exonic splicing enhancer ESE exonic splicing silencer ESS intronic splicing enhancer ISE intronic splicing silencer ISS
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A compact formalism, but…
score matrix
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Compression and reconstruction of motifs
AGG AGT CGT Experimental assessed binding sites zip AGG AGT CGG CGT A G consensus sequence G unzip C T
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Intron definition / exon definition
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Modello di exonic splicing enhancer mediato da proteine SR
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Modello di exonic splicing silencer
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elements promoting exons
elements promoting introns
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ESE, ISS: esone ESS, ISE: introne
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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 …
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EXPERIMENTALLY ASSESSED BINDING
Some comparisons among literature data (SpliceAid) and prediction tools SEQUENCE SPLICEAID COMPETING TOOLS EXPERIMENTALLY ASSESSED BINDING ESE Finder Rescue ESE Splicing Rainbow ACAAC YB-1 no binding no ESE SRp40 GAAGAAGA HTra2A, HTra2B1, SF2/ASF, SC35, SRp40, SRp55, SRp75 3 ESE Tra2B CUGGCGUCGUCGC SF2/ASF, SRp55 2 ESE SRp40, SRp55 UGACUG hnRNP A1 UUUUAGACAA hnRNP C1, Sam68, hnRNP A1, hnRNP D, 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 SRp55 hnRNP U
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Pan troglodytes average nucleotide divergence of just 1.2%
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Suggested papers Nature reviews. Genetics. 2002; 3(4): Listening to silence and understanding nonsense: exonic mutations that affect splicing. Cartegni L, Chew SL, Krainer AR. PMID: Nature reviews. Genetics. 2007; 8(10): Splicing in disease: disruption of the splicing code and the decoding machinery. Wang GS, Cooper TA. PMID:
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