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 f.piva@univpm.it

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

Genomic DNA mRNA ATGTAAACGTATATCGTGACAGTGGTCTGTTAGTATTCCTTTAGTCATGGTTT ATGTAAACTGGTCTGTTATCATGGTTT mRNA

attggaaaccgaaacccgttggtcacctctgcaatagccctccctccctcacttctacaattttgtgacagtggtcttgttttctgcattctctgcttcacgtgcttgttttgttggagcgcgtttgcatgctgctttaaattctgaaatattaaaaaaatttcgaagtttttcagcacatgggatgggagttttgaatttcaattttttaaaaacatttttctgtgattagtgccgtcgtggcacggctgttagccgcctatccggtttattcgatactttGTGAGTTTTTTGTAACTTTATGGTCGTCGAAATGGGAAAACTTGGCCACCAATATAAGTTTGGAAAACAATTTCCTAAAAATAAAATAATTGAACTTTTCCGATGAATAAAAAAATCGATCAGATATTCTGGAAAAAAAATCGATAAATTAATCGATTTTCTTGGAAAATACATCGAAAAATTGAGAAAAATAGAAAAATGAATGTTTTTCGATTACCGATTTATTGATTTTTCGTGAAAACTGAGTTCAGATAATTTTAAAAGCAATGTTTTTCATTTTTCAAATCAGAATCACTATAGTTTTGAAAAATCAATAATTAATTTATTGATTTTTCAATATAATTTTTTGGAAAAAATAGAAAAATCCCTTTCTAAAAGTTTTAAATTTCCAAGAAAAATTCATTTTCAAAATCACCAACGCGCTCTATAGAGTAGTCGATGAAAATCTCCGTTAAGGGTGCATGGGCAAAACGCGCTCGAACGACAATTGTTATTGTATGTTTGGTCTTGCAACGAAAAGTTTGAAAAATTGAAAAAAAGTTGTGTCTGATACATTTTTTTTTGGCATTTTCTGCTATTTTACACCAGAAAAAATTTAATAAACATAAAAAATCGAAATTTTTCAAGTTGGACAATTTTCAGtgagcatcttatccatcctagttctcagttcaggacttgtgcacattcgtttagagccagatattcgcaaagccttttcaccggatgattcagatgctggataGTAAGTGACTACTGACCTTGAAGCCTCCTTCCTCCACCAGTCAGAAATAACACGTTTTTTCGCAATGTTTTTCTTTTTCTAATTCGATTTCCCTTTCTCCCTTTCTTATTGTGATTTGGTCAATGTTTGGTTGACTGGGAAGAAAATTGAATTTTTTTGGAATTCCACTTGAAGTTAAAAAACCCAAAATAAATATTTGATCAAAAATAAATAAGAAAAAAAAGAAAACTTTAAAGCAAATGAAAATTTCGTTCGTAACTATTTTGTTAATTTTTTTAAAACTCCTATTTTAAATATATGCTTTTTGCGGAAATTTCTATAAATTTTTTTACATTTTTCAGtgaaacccgtgtctggctggaatactacggactcgacatctatccggaacgagcattctgtatttttaccgccaagcgcgaaaattccagtattctccaggaaggcgcactggcagacGTAAGTTGATTCTCCGTCACGCCCACTTTTCTGGCGGGAATTTAAAAAATTTCAGatttatactgtggacaatcgactatcggcggcagttggctaccaagatggggatggacgaaaaaattgcgatccactctgcgacttgaacagcccctttcacttgttagcgGTAGGTGGTGGTCTAGGGTGTCATTTTTCGATTTTTTCAATTATTCGATGTTTTTAGTGAAAATCGAAAAATCTAAAAATTGAAAATCGAAAAATGAAAGAAACATTGTTTTTTGGGGACCAAACATCTTAATGAATTTAACAACAGGGAAAACTGAACAGAAACCTGGACGGTCTTATCCCATTTATCTATATTCTTAAAATGAATGATGGAGAAAAAAGTTAAAATAAAAACATTATCAGCTTTTTGTAAGTTTTTCTCAAAAATTGTTCGATTTTTCGATTTTCTAAAAAGTCGAAAAACCGAAACCCTTGGTGGTGGTGGTGGTGGACTAGAAAACTCTTCAACGACCACATGGCAATTTTCAGaatttgacgcggagaaacaatggtaccacaagtgtattcacctatccggatatgccatatagcggactggatattttcctgggacttcacttgagtaatgcggattttggtaagattttttttgaaatgttaaatgaaaagttgaaaaatagtttttatgatttagccactttccagttaaaatttcatttttttaactataaaaagttctggaaaaatg

Struttura tipica dei geni umani esoni introni

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

Lo splicing avviene in tutto il trascritto, anche nelle zone non codificanti

R = G, A Y = T, C

= + + 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

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

introne (5’ss)

snRNP U1 Sm protein

RBD: RNA binding domain C5

snRNP U2 Sm protein si appaiano con snRNA U6 si appaia al sito di ramificazione

U17 U5

Lo splicing è tessuto specifico 1 2 3 5 Muscolo cardiaco 1 2 3 4 5 1 4 3 5 Muscolo uterino

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

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

5’splice sites

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

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

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

An additional exonic constraints: the splicing code

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

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 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):294-295.

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: 426-429 20 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 AGG AGT CGT Experimental assessed binding sites zip AGG AGT CGG CGT A G consensus sequence G unzip C T

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 …

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

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