Genome Analysis Research Group

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Genome Analysis Research Group
Leibniz Institute for Age Research Fritz
Lipmann Institute
Violating the splicing rulesTG dinucleotides
function as 3' splice sites in U2-dependent
introns
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subtle alternative splicing
overview
Cartegni et al., Nat Rev Genet 3, 285 (2002)
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subtle alternative splicing
Exceptional splice sites?
GNAS
adenylyl cyclase stimulatory G-protein G?S
Quan Forte MCB 10,910 (1990) Pollard et al. JBC
277,15241 (2002)
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subtle alternative splicing
Exceptional splice sites?
GU-AG rulefor U2-dependent introns
Cartegni et al., Nat Rev Genet 3, 285 (2002)
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subtle alternative splicing
Exceptional splice sites?
A/A
A/G
G/G
1
3
2
number of protein isoforms
Hiller et al., Am J Hum Genet 78, 291 (2006)
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subtle alternative splicing
Exceptional splice sites?
GNAS
adenylyl cyclase stimulatory G-protein G?S
NAGNAG
no
no SNP!
Quan Forte MCB 10,910 (1990) Pollard et al. JBC
277,15241 (2002)
unusual UGacceptor site!
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subtle alternative splicing
Exceptional splice sites?
Identification and validation of 36 TG acceptors
in the human genome
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mining spliced alignments
Approach
neutral approach to search for 3' splice
variants splice variant pairs (SVP)
spliced alignment of ESTs
3 splice site (E)
3 splice site (I)
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mining spliced alignments
Approach II
neutral approach to search for non-canonical
splice sites based on RefSeq transcripts
(independent of alternative splicing)
spliced alignment of RefSeq transcripts
UG 3 splice site
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mining spliced alignments
Pipeline
systematic in silico screening for unusual
alternative 3 splice sites of human introns
human EST-to-genome alignment
human RefSeq-to-genome alignment
quality filtering
BLAST-validation of EST-supported splice variants
manual inspection
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mining spliced alignments
Pipeline
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mining spliced alignments
Pipeline
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mining spliced alignments
ESTs vs. RefSeqs
RefSeq-based
EST-based
raw cases of unusual intron termini
44
39
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artifacts
30
34 (77)
considered valid
9 (23)
6
28
3
92 reproducible in RT-PCR
non-canonical TG 3'ss in U2 introns
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mining spliced alignments
Experimental validation
CNBP (cellular nucleic acid binding protein)
exon3
AG-derived splice variants
UG-derived splice variants
UG-derived splice variants were experimentally
validated!
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functional relevance
More than just noise?
noise produced by error-prone spliceosomes?
variants awaiting functionalization during
evolution?
functionally relevant alternative splicing?
Indicators
flanking sequence conservation
splice site conservation
tissue-specific splicing pattern
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functional relevance
intron sequence conservation
H.sapiens
conservation offlanking intron sequence
?
M.musculus
exon
intron
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functional relevance
intron sequence conservation
efficient TG alternative splicing under purifying
selection
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functional relevance
intron sequence conservation
RYK intron 7
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functional relevance
tissue-dependent splice patterns
CNBP
BRUNOL4
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structural and mechanistical implications
structural propertiesand mechanistic implications
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structural and mechanistical implications
structural pattern
Significant structural constraint observed
TGs were exclusively found as alternative
acceptors, part of TG-AG splice site tandems
6 AG-TG, up to ? 4 nt
31 TG-AG, up to ? 28 nt
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functional relevance
Mechanism of SS choice
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structural and mechanistical implications
Mechanism of SS choice
AG-AG tandems may have separate branchpoints
BP
AG
AG
BP
...
...
... allowing large-distance splice sites.
TG-AG tandems share the same branchpoint (BP)
BP
TG
AG
...
...
... limiting the distance of splice sites.
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functional relevance
Mechanism of SS choice
rare but high-confidence TG acceptors TG
acceptors always alternative TG-AG acceptor
distance constraint high conservation of
alternative splicing at TG acceptors
... specific mechanism?
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functional relevance
Mechanism of SS choice
splicing step I
intron recognition
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U2 snRNP
U2AF
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U2AF
typically 15-40 nt
...nnCUaAcnnnnnnnnnnnUGnnnnnnnnnAGNNNNNNNNN...
exon
Lallena et al. 2002, Cell 109,285
SPF45
...or another factor?
splicing step II
splice site selection
AG is required for intron recognition during
splicing step I
TG splice site selection takes place in splicing
step II
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Acknowledgments
Inst. Clin. Mol. Biol., Univ. Kiel
Stefan Schreiber Philip Rosenstiel
Bioinformatics, Univ. Freiburg
Michael Hiller Rolf Backofen
Genome Analysis, FLI Jena
Stefanie Schindler Stefan Taudien Klaus
Huse Niels Jahn Matthias Platzer