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)

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
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
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
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)
Sureau et al. (2001) EMBO J 20:1785-96

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)
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

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:
Alternatively spliced
EST, reading frame 1
Alternatively spliced
EST, reading frame 2
Alternative polyadenlyation signals are biased against recovery