17 dicembre 2010
Trento
Linguaggi sovrapposti: il codice dello splicing
Piva F, Giulietti M, Principato G
Department of Biochemistry, Biology and Genetic
Politechnic University of Marche, Ancona
One point
mutation at
a time
BRCA1 exon 18
100%
20%
17 18 19
80%
17 19
17 18 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
Complete exon 18 skipping
Decreased efficiency
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
pathological
pathological
100
90
80
70
60
50
40
30
20
10
0
5’-ACAGTTGTTGGCGGTTG-3’
TACCACCC TTATT
GGTTC AA CCGC
G G
T
pathological
% exon 9 inclusion
WT
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 149T150G 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
The genetic code is degenerate, but it is not all rodustness
. . . Ala Val
Arg . . .
GCA GTA CGA
C
C
C
G
G
G
T
T
T
AGA
G
4 * 4 * 6 = 96
Three AAs specified by 96
synonymous words
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
An additional exonic constraints: the splicing code
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
Disruption
of 5’ss
restores
normal
splicing
TAGataata
TAGgtggga
TAGgtaata
CAGgtattg
CAAgtattg
CAAgtaagc
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
20
WT: GGCCAGGTAAGTGATA
20
mutations
21
21
DEL: GGCCAG____GTGATA
MUT: GGCCAGGTCTGTGATA
M WT del mut
results
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
20 21
21
AIM:
mRNA
structure
pre mRNA
sequence
SPLICING PREDICTION TOOL
A compact formalism, but…
score
matrix
Compression and reconstruction of motifs
Experimental
assessed
binding
sites
AGG
AGT
CGT
AGG
CGT
zip
consensus
sequence
A G
G
C T
unzip
AGG
AGT
CGG
CGT
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 …
Some comparisons among literature data (SpliceAid) and prediction tools
SEQUENCE
ACAAC
SPLICEAID
EXPERIMENTALLY
ASSESSED BINDING
YB-1
PREDICTORS
ESE Finder
Rescue ESE
no binding
no ESE
Splicing
Rainbow
SRp40
3 ESE
Tra2B
GAAGAAGA
HTra2A, HTra2B1,
SF2/ASF, SC35,
no binding
SRp40, SRp55, SRp75
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,
no binding
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
no ESE
hnRNP U
SRp55
SpliceAid 2
Giunzioni esoniche in fase 0
m
-1 1
n
10
A……………………………………………………..A
T……………………………………………………...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
Giunzioni esoniche in fase 1
-10
m
-1 1
n
10
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
Giunzioni esoniche in fase 2
-10
m
-1 1
n
10
A………………………………....A
A…………………………………………..A
A……………………………………………A
A………T
A……………………….A
T……T
C…………...G
Correlazioni favorite
tra la fine di un esone e l’inizio del successivo
Esoni che iniziano in fase 0 e terminano in fase 0
1
10 -10
-1
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
Fase 0 1
1
10
-10
Fase 0 2
-1
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
-10
-1
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
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
ridondanti
Seq NON
ridondanti
Seq 1
Seq 2
Seq 3
Seq 4
Seq 5
…
Seq N
Seq
ridondanti
Seq NON
Ridondanti
Seq
ridondanti
Seq NON
Ridondanti
Seq 2
Seq 3
Seq 4
Seq 5
…
Seq N
Seq 1
Seq 3
Seq 4
Seq 5
…
Seq N
Seq 1
Seq 2
Seq
ridondanti
Seq NON
Ridondanti
Seq
ridondanti
Seq NON
Ridondanti
Seq 4
Seq 5
…
Seq N
Seq 1
Seq 2
Seq 3
Seq 5
…
Seq N
Seq 1
Seq 2
Seq 3
Seq 4
nallineamen ti 
N 1
x
x 1
N  ( N  1)
2
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 F
Assessing sediment hazard through a Weight Of Evidence approach with bioindicator organisms: a practical model to
elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays
Chemosphere 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 C
Searching for a relationship between the Serotonin Receptor 2A Gene variations and the development of Inward and Outward Personal
Meaning Organisations
Psychiatric Genetics 2010 accepted
Lavori inviati nel 2010:
Piva F, Giulietti M, Ballone Burini A, Principato G
SpliceAid 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 G
Bioinformatic analyses to select phenotype affecting polymorphisms in HTR2C gene
Piva F, Giulietti M, Principato G
CLIP 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 A
ADH4 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 P
TRAM (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
Giovanni
Principato
Francesco
Piva
Matteo
Giulietti