Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute...
-
Upload
loren-sutton -
Category
Documents
-
view
219 -
download
2
Transcript of Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute...
![Page 1: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/1.jpg)
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)
![Page 2: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/2.jpg)
subtle alternative splicingoverview
Cartegni et al., Nat Rev Genet 3, 285 (2002)
![Page 3: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/3.jpg)
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
![Page 4: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/4.jpg)
subtle alternative splicingExceptional splice sites?
Cartegni et al., Nat Rev Genet 3, 285 (2002)
GU-AG rulefor U2-dependent introns
![Page 5: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/5.jpg)
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
![Page 6: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/6.jpg)
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
![Page 7: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/7.jpg)
subtle alternative splicingExceptional splice sites?
Identification and validation of 36 TG acceptors in the human genome
![Page 8: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/8.jpg)
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”)
![Page 9: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/9.jpg)
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)
![Page 10: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/10.jpg)
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
![Page 11: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/11.jpg)
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
![Page 12: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/12.jpg)
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
![Page 13: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/13.jpg)
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
![Page 14: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/14.jpg)
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 :
![Page 15: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/15.jpg)
functional relevanceintron sequence conservation
conservation offlanking intron sequence
H.sapiens
M.musculus?
exonintron
![Page 16: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/16.jpg)
functional relevanceintron sequence conservation
efficient TG alternative splicing under purifying selection
![Page 17: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/17.jpg)
functional relevanceintron sequence conservation
RYK intron 7
![Page 18: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/18.jpg)
functional relevancetissue-dependent splice patterns
CNBPBRUNOL4
![Page 19: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/19.jpg)
structural and mechanistical implications
structural propertiesand mechanistic implications
![Page 20: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/20.jpg)
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
![Page 21: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/21.jpg)
functional relevanceMechanism of SS choice
![Page 22: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/22.jpg)
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.
![Page 23: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/23.jpg)
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?
![Page 24: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/24.jpg)
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
![Page 25: Genome Analysis Research Group Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules: TG dinucleotides function as.](https://reader035.fdocuments.in/reader035/viewer/2022062516/56649e425503460f94b3530f/html5/thumbnails/25.jpg)
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