Alternative Splicing

1
Alternative Splicing
Genomic DNA Sequence
Transcription
pre-mRNA
Intron
Intron
Intron
Exon
Exon
Exon
Exon
Exon
RNA Processing
Gm
AAAAA
Gm
AAAAA
mRNA
mRNA
2
Alternative Splicing Data Sources are Large and
Growing
Curated databases SWISS-PROT and RefSeq both
support annotation of experimentally supported
alternative splicing
cDNA Sequencing Projects RIKEN sequenced gt21000
full length mouse cDNAs Many other projects
underway (human, fly, plants,)
Shinagawa et al. (2001) Nature 409685-90
Microarray detection Direct or indirect
alternative splicing detection
Hu et al. (2001)Genome Res 111237-45 Yeakley et
al. (2002) Nat Biotech 20353-9
Public EST data sources (dbEST) gt4.5 million
human EST sequences gt12 million total EST
sequences About 1000 new sequences per day
Boguski et al. (1993) Nat Gen 4332-3
3
Nonsense-Mediated mRNA Decay
Genomic DNA
pre-mRNA
Exon
Intron
Exon
Exon
Intron
Exon junction complex
mRNA
Gm
AAAAAAAAA
Termination codon is on the last exon (not
premature)
Leeds et al. (1991) Genes Dev 52303-14
Nagy and Maquat (1998) TIBS 23198-9
Le Hir et al. (2000) Genes Dev 141098-1108
Mitchell and Tollervey (2001) Curr Opin Cell Biol
13320-5
Ishigaki et al. (2001) Cell 106607-17
Lykke-Andersen et al. (2001) Science 2931836-9
Kim et al. (2001) EMBO 202062-68
4
Nonsense-Mediated mRNA Decay
Interaction between EJC and release factors
triggers NMD
Decapping and degradation
Gm
mRNA
AAAAAAAAA
Termination codon gt 50nt before last exon
junction (Premature Termination Codon)
Leeds et al. (1991) Genes Dev 52303-14
Nagy and Maquat (1998) TIBS 23198-9
Le Hir et al. (2000) Genes Dev 141098-1108
Mitchell and Tollervey (2001) Curr Opin Cell Biol
13320-5
Ishigaki et al. (2001) Cell 106607-17
Lykke-Andersen et al. (2001) Science 2931836-9
Kim et al. (2001) EMBO 202062-68
5
Nonsense-Mediated mRNA Decay
Translated normally
ORF
Gm
mRNA
AAAAAAAAA
Degraded by NMD
ORF
Gm
mRNA
AAAAAAAAA
gt50 nt
6
NMD is Pervasive
NMD is a critical process in normal cellular
developement Wagner and Lykke-Andersen (2002) J
Cell Sci 1153033-8
V(D)J recombination
Wang et al. (2002) J Biol Chem 27718489-93
Renders recessive many otherwise dominant
mutations
Cali and Anderson (1998) Mol Gen Genet 260176-84
7
NMD is Pervasive
V(D)J recombination
NMD is a critical process in normal cellular
developement Wagner and Lykke-Andersen (2002) J
Cell Sci 1153033-8
Wang et al. (2002) J Biol Chem 27718489-93
Renders recessive many otherwise dominant
mutations
Cali and Anderson (1998) Mol Gen Genet 260176-84
8
Transcriptional Regulation
Gene locus
transcription
pre-mRNA
productive splicing
productive mRNA
RUST
translation
Protein
9
Transcriptional Regulation
Transcriptional Regulation
RUST
Gene locus
Gene locus
transcription
pre-mRNA
pre-mRNA
productive splicing
Productive mRNA
Productive mRNA
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Alternative Splicing Can Yield Isoforms
Differentially Subjected to NMD
Nucleus
Nucleus
DNA
DNA
pre-mRNA
pre-mRNA
mRNA
mRNA
Premature termination codon
NMD
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SC35 Auto-regulation
SC35 Locus
transcription
SC35 pre-mRNA
splicing
alternative splicing
Productive SC35 mRNA
translation
SC35 protein
Sureau et al. (2001) EMBO J 201785-96
12
SC35 Auto-regulationAlternative splicing coupled
with nonsense-mediated decay
SC35 Locus
SC35 pre-mRNA
Productive SC35 mRNA
SC35 protein
ORF
SC35 pre-mRNA
Gm
AAAAA
SC35 mRNA
SC35 protein
SC35 pre-mRNA
SC35 mRNA (with premature termination codon)
Gm
AAAAA
Sureau et al. (2001) EMBO J 201785-96
13
SC35 Auto-regulationAlternative splicing coupled
with nonsense-mediated decay
Gm
Gm
Sureau et al. (2001) EMBO J 201785-96
14
EST-inferred human isoforms
0
10000
2000
4000
6000
8000
NMD Candidates
1989 (35 of 5693)
5693
Alternative isoforms
All isoforms, including canonical
8820
15
Inferred Isoforms
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
NMD Candidates
2078 (34 of 5970)
5970
Alternative isoforms
9282
All isoforms, including canonical
16
Canonical Splice Forms
Genomic DNA Sequence
Exon 1
Exon 3
Exon 2
Exon 4
Refseq-coding gene
mRNA
17
gt92 identity, gaps allowed
Aligned EST 5 end does not indicate reading
frame
18
Premature Stop Codons
19
Alternative Isoform Inference from Splice Pairs
20
Alternative Splice Pairs, by Mode
21
Alternative Splice Pairs, by Mode
22
Splice Pairs Generating Premature Stops
23
EST coverage and premature stops
For 76 of isoforms with premature stops
ESTs cover a PTC splice junction downstream
In 80 of these isoforms, there is a PTC in every
reading frame
Alternative polyadenlyation signals are biased
against recovery
24
EST coverage and premature stops
For 76 of isoforms with premature stops
RefSeq mRNA
Alternatively spliced EST, reading frame 0
ESTs cover a PTC splice junction downstream
In 80 of these isoforms, there is a PTC in every
reading frame
Alternatively spliced EST, reading frame 1
Alternatively spliced EST, reading frame 2
Alternative polyadenlyation signals are biased
against recovery
25
Alternative Polyadenylation?
once every 3115 nucleotides
Polyadq
RefSeq mRNA
Tabaska and Zhang. (1999) Gene 231 77-86
once every 2560 nucleotides
NMD-candidate alternatively spliced EST
26
Alternative Splicing in Human
0
10000
2000
4000
6000
8000
NMD Candidates
2078 (35 of 5970)
5970
Alternative isoforms
9527
All isoforms, including canonical
27

Previous and new RUST targets
Class Experimental Evidence Among Our Results
Splicing Factors AUF1, SC35 SRP20, SRP30b (in C. elegans) Sureau et al. (2001) EMBO J 201785-96 Wilson et al. (1999) Mol Cell Bio 194056-64 Morrison et al. (1997) PNAS 949782-9785 AUF1, 10 new
Ribosomal Proteins L3, L7a, L10a, L12 (in C. elegans) L30, S14B (in S. cerevisiae) Mitrovich Anderson (2000) GenesDev 142173-84 L3, L7a, L10a, L12, 11 new

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Alternative Splicing
Genomic DNA Sequence
Transcription
pre-mRNA
Intron
Intron
Intron
Exon
Exon
Exon
Exon
Exon
Exon
Gm
AAAAA
Gm
AAAAA
mRNA
mRNA
29
Alternative Splicing
Recruitment of Sequence. Deletion of Sequence.
Frameshift and Truncation.
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Premature Stop Codons
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EST Limitations
Single pass sequencing errors
Incompletely processed transcripts
3 end bias
Library contamination
Thanaraj (1999) NAR 272627-37
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Alternative Splicing EST Analysis
From data in Brett et al. (2000) FEBS Lett
47483-6
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Alternative Isoform Inference from Splice Pairs
34
Alternative Splice Pairs, by Mode
35
Alternative Splice Pairs, by Mode
36
Splice Pairs Generating Premature Stops
37
EST coverage and premature stops
For 76 of isoforms with premature stops
RefSeq mRNA
Alternatively spliced EST, reading frame 0
ESTs cover a PTC splice junction downstream
In 80 of these isoforms, there is a PTC in every
reading frame
Alternatively spliced EST, reading frame 1
Alternatively spliced EST, reading frame 2
Alternative polyadenlyation signals are biased
against recovery
38
(No Transcript)