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
Shinagawa et
al. (2001) Nature
409:685-90 underway (human, fly,
Many
other
projects
plants,)
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)
Boguski et al. (1993) Nat

Gen 4:332-3
>4.5 million human EST sequences

>12 million total EST sequences
Nonsense-Mediated mRNA Decay

Genomic DNA
pre-mRNA
Intron
Gm
mRNA
Exon
Exon
Intron
Exon
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)

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

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

Translated normally
ORF
mRNA
AAAAAAAAA
Gm
Degraded by NMD
ORF
Gm
mRNA
AAAAAAAAA
>50
n
NMD is Pervasive
1498 of 1500 genes surveyed from
fungi, plants, insects and
vertebrates obey the PTC rule
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
35
C
S
5
3
SC
5
3
C
S
SC35 mRNA
SC35
protein
SC35 pre-mRNA
Gm
SC
35
SC
35
AAAAA
SC35 mRNA
AAAAA (with premature
termination codon)
EST-inferred human isoforms
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)
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
Alternative Splicing EST Analysis
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:
Alternative polyadenlyation signals are biased against recovery

Alternative Splicing

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Alternative Splicing

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Alternative Splicing

Genomic DNA Sequence

Alternative Splicing Data

cDNA Sequencing Projects

Direct or indirect alternative splicing detection

Public EST data sources

Boguski et al. (1993) Nat

>4.5 million human EST sequences

Nonsense-Mediated mRNA Decay

Exon junction complex

Leeds et al. (1991) Genes Dev 5:2303-14

Nonsense-Mediated mRNA Decay

Termination codon > 50nt before

Leeds et al. (1991) Genes Dev 5:2303-14

Nonsense-Mediated mRNA Decay

4.3% of reviewed RefSeqs have PTCs

NMD is a critical process in

Renders recessive many otherwise

Alternative Splicing Can Yield Isoforms

Productive SC35 mRNA

EST-inferred human isoforms

1989 (35 % of 5693)

Canonical Splice Forms

Pruitt, K.D. et al (2001)

Lander et al. (2001) Nature 409:

Genomic DNA Sequence

Wheelan et al. (2001)

Construct genes from

ESTs from dbEST

Boguski et al., (1993) Nat Genet 4, 332-3.

Cluster ESTs w/ WU-BLAST2

Align ESTs w/ sim4

Alternative Isoforms of Refseq-coding genes

Previous and new RUST targets

L3, L7a, L10a, L12 (in C. elegans)

Among Our Results

AUF1, *10 new

Premature Stop Codons

Alternative Splicing EST Analysis

From data in Brett et al. (2000) FEBS Lett 474:83-6

Alternative Isoform Inference

Alternative Splice Pairs, by Mode

Alternative Splice Pairs, by Mode

Splice Pairs Generating Premature Stops

EST coverage and premature stops

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