Genome Analysis Research GroupLeibniz Institute for Age Research – Fritz Lipmann Institute
Violating the splicing rules:TG dinucleotides function as 3' splice
sites in U2-dependent introns
Karol Szafranskipresented by Rileen Sinha
to appear soon in Genome Biology 8 (2007)
subtle alternative splicingoverview
Cartegni et al., Nat Rev Genet 3, 285 (2002)
subtle alternative splicingExceptional splice sites?
Quan & Forte MCB 10,910 (1990)Pollard et al. JBC 277,15241 (2002)
...UGGAGA gu.....uuacug cag
...UGGAGA gu.....uuacug CAG UGAGAA...
UGAGAA...
adenylyl cyclase stimulatory G-protein GS
GNAS
subtle alternative splicingExceptional splice sites?
Cartegni et al., Nat Rev Genet 3, 285 (2002)
GU-AG rulefor U2-dependent introns
subtle alternative splicingExceptional splice sites?
`
G/G A/G A/A
1 3 2
number of protein isoforms
Hiller et al., Am J Hum Genet 78, 291 (2006)
genotype
subtle alternative splicingExceptional splice sites?
adenylyl cyclase stimulatory G-protein GS
unusual UGacceptor site!
Quan & Forte MCB 10,910 (1990)Pollard et al. JBC 277,15241 (2002)
...UGGAGA gu.....uuacug cag
...UGGAGA gu.....uuacug CAG UGAGAA...
UGAGAA...
NAGNAGno SNP!
no
GNAS
subtle alternative splicingExceptional splice sites?
Identification and validation of 36 TG acceptors in the human genome
mining spliced alignmentsApproach
...NNNNNN nn.....nnnnnn nnn
...NNNNNN nn.....nnnnnn NNN NNNNNN...
NNNNNN...
neutral approach to search for 3' splice variants:splice variant pairs (SVP)
spliced alignment of ESTs:
3’ splice site (“E”)
3’ splice site (“I”)
mining spliced alignmentsApproach II
...NNNNNN nnn.....nnnnnnnug NNNNNN...
spliced alignment of RefSeq transcripts:
UG 3’ splice site
neutral approach to search for non-canonical splice sitesbased on RefSeq transcripts (independent of alternative splicing)
mining spliced alignmentsPipeline
systematic in silico screeningfor 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
mining spliced alignmentsPipeline
filter for paralogous gene loci
validate putative splice variants by explicit BLAST searches;choose only high-quality BLAST alignments around the exon-exon junctionrequire at least two ESTs as evidence for a splice variant
quality aspects of EST-based screen for splice variants
mining spliced alignmentsESTs vs. RefSeqs
RefSeq-basedEST-based
44raw cases of unusual intron termini
39
8artifacts 30
34 (77%)considered valid 9 (23%)
6 328
non-canonical TG 3'ss in U2 introns
92% reproducible in RT-PCR
mining spliced alignmentsExperimental validation
CNBP (cellular nucleic acid binding protein)
UG-derived splice variants were experimentally validated!
AG-derived splice
variants
UG-derived splice
variants
exon3 exon4
functional relevanceMore than just noise?
noise produced by error-prone spliceosomes?
variants awaiting functionalization during evolution?
functionally relevant alternative splicing?
flanking sequence conservation
splice site conservation
tissue-specific splicing pattern
Indicators :
functional relevanceintron sequence conservation
conservation offlanking intron sequence
H.sapiens
M.musculus?
exonintron
functional relevanceintron sequence conservation
efficient TG alternative splicing under purifying selection
functional relevanceintron sequence conservation
RYK intron 7
functional relevancetissue-dependent splice patterns
CNBPBRUNOL4
structural and mechanistical implications
structural propertiesand mechanistic implications
structural and mechanistical implications
TGs were exclusively found as alternative acceptors, part of TG-AG splice site tandems
structural pattern
Significant structural constraint observed:
6 AG-TG, up to 4 nt
31 TG-AG, up to 28 nt
functional relevanceMechanism of SS choice
structural and mechanistical implicationsMechanism of SS choice
AG-AG tandems may have separate branchpoints
BPAG AG... ...
... allowing large-distance splice sites.
BP
TG-AG tandems share the same branchpoint (BP)
BP TG AG... ...
... limiting the distance of splice sites.
functional relevanceMechanism 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?
functional relevanceMechanism of SS choice
...nnCUaAcnnnnnnnnnnnUGnnnnnnnnnAGNNNNNNNNN...
U2AF35
exon
U2 snRNP
typically 15-40 nt
U2AF65
splicing step IIsplice site selection
TG splice site selection takes place in splicing step II
AG is required for intron recognition during splicing step I
intron recognitionsplicing step I
SPF45...or another factor?
Lallena et al. 2002, Cell 109,285
Acknowledgments
Stefanie SchindlerStefan TaudienKlaus HuseNiels JahnMatthias Platzer
Genome Analysis, FLI Jena
Michael HillerRolf Backofen
Bioinformatics, Univ. Freiburg
Stefan SchreiberPhilip Rosenstiel
Inst. Clin. Mol. Biol., Univ. Kiel
