Download Alternative Splicing

Alternative Splicing
Genomic DNA Sequence
Transcription
pre-mRNA
Intron
Exon
Exon
Exon
Intron
Exon
Intron
Exon
RNA Processing
Gm
mRNA
Gm
AAAAA
AAAAA
mRNA
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 >21000 full length mouse cDNAs
Many other projects underway (human, fly, plants,…)
Shinagawa et al. (2001) Nature 409:685-90
Microarray detection
Direct or indirect alternative splicing detection
Hu et al. (2001)Genome Res 11:1237-45
Yeakley et al. (2002) Nat Biotech 20:353-9
Public EST data sources (dbEST)
>4.5 million human EST sequences
>12 million total EST sequences
About 1000 new sequences per day
Boguski et al. (1993) Nat
Gen 4:332-3
Nonsense-Mediated mRNA Decay
Genomic DNA
pre-mRNA
Gm
mRNA
Exon junction complex
AAAAAAAAA
Leeds et al. (1991) Genes Dev 5:2303-14
Nagy and Maquat (1998) TIBS 23:198-9
Le Hir et al. (2000) Genes & Dev 14:1098-1108
Mitchell and Tollervey (2001) Curr Opin Cell Biol 13:320-5
Ishigaki et al. (2001) Cell 106:607-17
Lykke-Andersen et al. (2001) Science 293:1836-9
Kim et al. (2001) EMBO 20:2062-68
Termination codon is
on the last exon
(not premature)
Nonsense-Mediated mRNA Decay
Interaction between EJC and
release factors triggers NMD
Decapping and degradation
Gm
mRNA
AAAAAAAAA
Termination codon > 50nt before
last exon junction
(Premature Termination Codon)
Leeds et al. (1991) Genes Dev 5:2303-14
Nagy and Maquat (1998) TIBS 23:198-9
Le Hir et al. (2000) Genes & Dev 14:1098-1108
Mitchell and Tollervey (2001) Curr Opin Cell Biol 13:320-5
Ishigaki et al. (2001) Cell 106:607-17
Lykke-Andersen et al. (2001) Science 293:1836-9
Kim et al. (2001) EMBO 20:2062-68
Nonsense-Mediated mRNA Decay
Translated normally
Gm
AAAAAAAAA
Degraded by NMD
Gm
AAAAAAAAA
NMD is Pervasive
1498 of 1500 genes surveyed from
fungi, plants, insects and
vertebrates obey the PTC rule
Nagy and Maquat (1998) TIBS 23:198-9
4.3% of reviewed RefSeqs have PTCs
34% have start codon after first exon
V(D)J recombination
Wang et al. (2002) J Biol Chem
277:18489-93
“NMD is a critical process in
normal cellular developement”
Wagner and Lykke-Andersen (2002) J Cell Sci 115:3033-8
Renders recessive many otherwise
dominant mutations
Cali and Anderson (1998) Mol Gen Genet 260:176-84
Transcriptional
Regulation
Gene locus
transcription
pre-mRNA
productive
splicing
productive
mRNA
RUST
translation
Protein
Transcriptional
Regulation
RUST
Gene locus
Gene locus
transcription
pre-mRNA
pre-mRNA
productive
splicing
Productive
mRNA
Productive
mRNA
Alternative Splicing Can Yield Isoforms
Differentially
Subjected to Nucleus
NMD
Nucleus
DNA
pre-mRNA
mRNA
DNA
pre-mRNA
mRNA
Premature
termination
codon
NMD
SC35 Auto-regulation
SC35 Locus
transcription
SC35 pre-mRNA
alternative
splicing
splicing
Productive SC35 mRNA
translation
SC35 protein
Sureau et al. (2001) EMBO J 20:1785-96
SC35 Locus
SC35 pre-mRNA
Productive SC35 mRNA
SC35 protein
SC35 Auto-regulation
Alternative splicing coupled with
nonsense-mediated decay
ORF
SC35 pre-mRNA
Gm
AAAAA SC35 mRNA
SC35
protein
SC35 pre-mRNA
SC35 mRNA
AAAAA (with premature
termination codon)
Sureau et al. (2001) EMBO J 20:1785-96
Gm
EST-inferred human isoforms
0
2000
NMD
Candidates
4000
6000
8000
10000
1989 (35 % of 5693)
Alternative isoforms
All isoforms, including canonical
5693
8820
Canonical Splice Forms
Refseq
mRNAs
Pruitt, K.D. et al (2001)
NAR 29: 137-40
Extract coding
regions
Lander et al. (2001) Nature 409:
860-921
Coding
Refseqs
Genomic DNA Sequence
Genomic
Contigs
Association
via LocusLink
Exon 1
Exon 2
Exon 3
Exon 4
Refseq-Contig Pairs
align w/
Spidey
≥98% id,
no gaps
Wheelan et al. (2001)
Gen Res 11:1952-7
Refseq-coding gene
mRNA
Construct genes from
aligned Refseq exons
& intervening genomic
introns (overlap
choose mRNA w/ largest
number of exons)
Refseq-coding genes
Refseq-coding genes
ESTs from dbEST
Boguski et al., (1993) Nat Genet 4, 332-3.
Cluster ESTs w/ WU-BLAST2
≥92% id, allow gaps
Florea, et al.,(1998)
Gen Res 8, 967-74.
Kan, et al. (2001)
Gen Res 11, 889-900.
Gish,(2002)(Wash.Univ.)
Align ESTs w/ sim4
Use TAP to infer
alternative mRNAs
Identification of
Alternative Isoforms
Alternative Isoforms of Refseq-coding genes
>92%
identity,
gaps
allowed
Aligned EST 5’ end
does not indicate
reading frame
Previous and new RUST targets
Class
Splicing
Factors
Ribosomal
Proteins
Experimental Evidence
AUF1, SC35
SRP20, SRP30b (in C. elegans)
Sureau et al. (2001) EMBO J 20:1785-96
Wilson et al. (1999) Mol Cell Bio 19:4056-64
Morrison et al. (1997) PNAS 94:9782-9785
L3, L7a, L10a, L12 (in C. elegans)
L30, S14B (in S. cerevisiae)
Mitrovich & Anderson (2000) GenesDev 14:2173-84
Among Our Results
AUF1, *10 new
L3, L7a, L10a,
L12, *11 new
Alternative Splicing
not
integer #
codons
Recruitment of
Sequence.
Deletion of
Sequence.
*Frameshift and
Truncation.
Premature Stop Codons
EST Limitations
Single pass
sequencing errors
Incompletely
processed transcripts
3’ end bias
Library
contamination
Thanaraj (1999) NAR 27:2627-37
Number of EST splice forms
Alternative Splicing EST Analysis
2000
1500
1000
500
0
Did not affect
Inserted stop
Changed
reading frame
codon
reading frame
From data in Brett et al. (2000) FEBS Lett 474:83-6
Alternative Isoform Inference
from Splice Pairs
Alternative Splice Pairs, by Mode
Alternative Splice Pairs, by Mode
Splice Pairs Generating Premature Stops
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