17 dicembre 2010Trento

Linguaggi sovrapposti: il codice dello splicing

Piva F, Giulietti M, Principato G

Department of Biochemistry, Biology and GeneticPolitechnic University of Marche, Ancona

Binding of DAZAP1 and hnRNPA1/A2 to an Exonic Splicing Silencer in a Natural BRCA1 Exon 18 MutantGoina E, Skoko N, Pagani F. Mol Cell Biol 2008; 28: 3850–3860

One point mutation at a time

BRCA1 exon 18

17 18 19

17 19

20%

80%17 18 19

100%

Two point mutations at a time

BRCA1 exon 18

Binding of DAZAP1 and hnRNPA1/A2 to an Exonic Splicing Silencer in a Natural BRCA1 Exon 18 MutantGoina E, Skoko N, Pagani F. Mol Cell Biol 2008; 28: 3850–3860

Complete exon 18 skipping Decreased efficiency

WT 5’-ACAGTTGTTGGCGGTTG-3’TACCACCC TTATTGGTTC AA CCGC G G T

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

Effect of variations 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

A

pathological

path

olog

ical

path

olog

ical

The genetic code is degenerate, but it is not all rodustness

. . . Ala Val Arg . . .

GCA C G T

GTA C G T

CGA C G TAGA G

GCAGTACGAGCAGTACGCGCAGTACGGGCAGTACGTGCAGTAAGAGCAGTAAGGGCAGTCCGAGCAGTCCGCGCAGTCCGGGCAGTCCGTGCAGTCAGAGCAGTCAGGGCAGTGCGAGCAGTGCGCGCAGTGCGGGCAGTGCGTGCAGTGAGAGCAGTGAGGGCAGTTCGAGCAGTTCGCGCAGTTCGGGCAGTTCGTGCAGTTAGAGCAGTTAGG

GCCGTACGAGCCGTACGCGCCGTACGGGCCGTACGTGCCGTAAGAGCCGTAAGGGCCGTCCGAGCCGTCCGCGCCGTCCGGGCCGTCCGTGCCGTCAGAGCCGTCAGGGCCGTGCGAGCCGTGCGCGCCGTGCGGGCCGTGCGTGCCGTGAGAGCCGTGAGGGCCGTTCGAGCCGTTCGCGCCGTTCGGGCCGTTCGTGCCGTTAGAGCCGTTAGG

GCGGTACGAGCGGTACGCGCGGTACGGGCGGTACGTGCGGTAAGAGCGGTAAGGGCGGTCCGAGCGGTCCGCGCGGTCCGGGCGGTCCGTGCGGTCAGAGCGGTCAGGGCGGTGCGAGCGGTGCGCGCGGTGCGGGCGGTGCGTGCGGTGAGAGCGGTGAGGGCGGTTCGAGCGGTTCGCGCGGTTCGGGCGGTTCGTGCGGTTAGAGCGGTTAGG

GCTGTACGAGCTGTACGCGCTGTACGGGCTGTACGTGCTGTAAGAGCTGTAAGGGCTGTCCGAGCTGTCCGCGCTGTCCGGGCTGTCCGTGCTGTCAGAGCTGTCAGGGCTGTGCGAGCTGTGCGCGCTGTGCGGGCTGTGCGTGCTGTGAGAGCTGTGAGGGCTGTTCGAGCTGTTCGCGCTGTTCGGGCTGTTCGTGCTGTTAGAGCTGTTAGG

4 * 4 * 6 = 96Three AAs specified by 96 synonymous words

An additional exonic constraints: the splicing code

exon31cryptic exonNF1 gene

A>G

ttttatagTGAGAATA

WT MUT

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.

La mutazione attiva un esone criptico (in rosso)

TAGgtaataTAGgtggga

TAGataataCAGgtattg

CAAgtattgCAAgtaagc

CAAgtaagg

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.

exon31cryptic exonNF1 gene

Disruption of 5’ss restores normal splicing

La seq 2 ha un sito di splicing in 5’ più debole della seq 1. La seq 3 non ha il sito.

ATM gene structure

20 21

M WT del mut

20 21

20 21

mutations

results

A new type of mutation causes a splicing defect in ATMPagani F, Buratti E, Stuani C, Bendix R, Dörk T, Baralle FENature Genetics 2002, 30: 426-429

20 21

WT: GGCCAGGTAAGTGATA

DEL: GGCCAG____GTGATA

MUT: GGCCAGGTCTGTGATA

AIM:

SPLICING PREDICTION TOOL

pre mRNAsequence

mRNAstructure

A compact formalism, but…

scorematrix

Experimental assessed binding sites

ACG

TGconsensus

sequence

AGGAGTCGGCGT

unzip

AGGAGTCGTAGGCGT

Compression and reconstruction of motifs

zip

elements promoting exons

elements promoting introns

ESE, ISS: esone

ESS, ISE: introne

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 …

PROTEINS REGULATING SPLICING STORED IN SPLICEAID

SEQUENCESPLICEAID PREDICTORS

EXPERIMENTALLY ASSESSED BINDING ESE Finder Rescue ESE Splicing

Rainbow ACAAC YB-1 no binding no ESE SRp40

GAAGAAGAHTra2A, HTra2B1, SF2/ASF, SC35, SRp40, SRp55, SRp75

no binding 3 ESE Tra2B

CUGGCGUCGUCGC no binding SF2/ASF, SRp55 2 ESE SRp40, SRp55

UGACUG hnRNP A1 no binding no ESE SRp40, SRp55

UUUUAGACAA

hnRNP C1, Sam68, hnRNP A1, hnRNP D, hnRNP E1, hnRNP E2, SRp38

no binding 1 ESE

hnRNP A2/B1, hnRNP C1/C2, hnRNP E1/E2, SRp40, SRp55, U2AF65

UGUGUGUGUGUGUGUGUG CUG-BP1, ETR-3, TDP43 SRp55 no ESE hnRNP U

Some comparisons among literature data (SpliceAid) and prediction tools

SpliceAid 2

-1 1 10m n

Giunzioni esoniche in fase 0

A……………………………………………………..AT……………………………………………………...T T…………………………T G……………………………G G………………………..G G…………………………………………..C G………………………………………………..G A…………………………………A A………………………………………….A A………………………….A A………………………………......A C………G C……………….C T…...G A………A C………G T………T A………………A C………………C A……………………….A A……………………….T C……………………….C C………………………….C

-10 -1 1 10m n

Giunzioni esoniche in fase 1

G………………………………………………..…..G A………………………………....A A.................................................T T……………………………………………..T A……………….A A…………………………A A…………………………………..A C......C C……….C G………C A..T C..C T…C T…T C……G T……T T………G C………….G C………………………..T C……………………………G

-10 -1 10m n1

Giunzioni esoniche in fase 2

A………………………………....A A…………………………………………..A A……………………………………………A A………T A……………………….A T……T C…………...G

Correlazioni favoritetra la fine di un esone e l’inizio del successivo

1 10 -1-10

G…………………………………..G G……………………………………..G G………………………………………………………...C G…………………………………………………………...C G…………………………………………………….……..T A……………………………A T…………………………….A T…………………………….T A…………………………………….A T……………………………………..T A………………………………................A T.................................................A T.................................................T T...........................................................T A………………….A T…………………..A A……………………..……A A........................................A A.................................................T T..................................................T C.......................G C....................G A...........A T............A T............T A.....................A T......................T A...............................A T................................T C...........C T...........T G….......G G…..........G

1 10 -1-10

T……………………………………..A T…………………………………..…...G T………………………………………….……….A T………………………………………...........................C C................................................................C A.......................................A T.......................................A A................................................A A................................................T T…………………………………..………….A T.................................................T C............................................................C T............................................................T T..................................A A..........................A T..........................A A....................................A C.................G G.......................................G A.................T T.................T A……………….……….A C……………………………………C

1 10 -1-10

T…………………………………………………………………….C A....................................A T....................................A A..............................................A T..............................................A C.....................................................C A.........................................................A A……………………………………………...........T A.............................................................C A........................A T........................A A..................................A T...................................T A.............................................A T..............................................T G..........................................T T.............A A.......................A A.......................T A.................................T T................................A A.........................................C G.....................................C

Fase 0 1 Fase 0 2Esoni che iniziano in fase 0 e terminano in fase 0

Correlazioni tra l’inizio e la fine degli introni umani

Elaborazioni in corso al CASPUR tramite ClustalW multiprocessore e programmazione multithreading… per ripetere le analisi su un insieme di geni con minore ridondanza

Seq ridondantiSeq 1Seq 2Seq 3Seq 4Seq 5…Seq N

Seq NON ridondanti

Seq ridondanti-Seq 2Seq 3Seq 4Seq 5…Seq N

Seq NON RidondantiSeq 1

Seq ridondanti--Seq 3Seq 4Seq 5…Seq N

Seq NON RidondantiSeq 1Seq 2

Seq ridondanti---Seq 4Seq 5…Seq N

Seq NON RidondantiSeq 1Seq 2Seq 3

Seq ridondanti----Seq 5…Seq N

Seq NON RidondantiSeq 1Seq 2Seq 3Seq 4

2

)1(1

1

NNx

tiallineamennN

x

Partendo da un insieme di 10.000 sequenze, se non effettuo nessun pruning,dovrei compiere al massimo 49.995.000 allineamenti

Altri lavori pubblicati o accettati nel 2010:

Piva F, Giulietti M, Nardi B, Bellantuono C, Principato G.An improved in silico selection of phenotype affecting polymorphisms in SLC6A4, HTR1A and HTR2A genes.Human Psychopharmacology 2010; 25: 153-61.

Piva F, Ciaprini F, Onorati F, Benedetti M, Fattorini D, Ausili A, Regoli FAssessing sediment hazard through a Weight Of Evidence approach with bioindicator organisms: a practical model toelaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassaysChemosphere 2010 accepted

Bianchi F, Raponi M, Piva F, Viel A, Bearzi I, Galizia E, Bracci R, Belvederesi L, Loretelli C, Brugiati C, Corradini F, Baralle D, Cellerino R.An intronic mutation in MLH1 associated with familial colon and breast cancer.Familial Cancer 2010 published

Nardi B, Turchi C, Piva F, Giulietti M, Castellucci G, Arimatea E, Rocchetti D, Rocchetti G, Principato G, Tagliabracci A, Bellantuono CSearching for a relationship between the Serotonin Receptor 2A Gene variations and the development of Inward and Outward Personal Meaning OrganisationsPsychiatric Genetics 2010 accepted

Lavori inviati nel 2010:Piva F, Giulietti M, Ballone Burini A, Principato GSpliceAid 2: a database of human splicing factors expression data and RNA target motifs

Piva F, Giulietti M, Baldelli L, Nardi B, Bellantuono C, Armeni T, Saccucci F, Principato GBioinformatic analyses to select phenotype affecting polymorphisms in HTR2C gene

Piva F, Giulietti M, Principato GCLIP data to detect polymorphisms lying in splicing regulatory motifs: a method to refine SNP selection in association studies

Turchi C, Piva F, Solito G, Principato G, Buscemi L, Tagliabracci AADH4 intronic variations are associated with alcohol dependence: results from an Italian case-control association study

Lenzi L, Facchin F, Piva F, Giulietti M, Pelleri MC, Frabetti F, Vitale L, Casadei R, Canaider S, Bortoluzzi S, Coppe A, Danieli GA, Principato G, Ferrari S, Strippoli PTRAM (Transcriptome Mapper): database-driven creation and analysis of transcriptome maps from multiple sources

Facchin F, …, Piva F,....Complexity of bidirectional transcription and alternative splicing at human RCAN3 locus

GiovanniPrincipato

FrancescoPiva

MatteoGiulietti