Alternative SplicingGenomic DNA Sequence

Gm

AAAAA

Exon ExonIntron Intron Intron

Exon ExonExon

Gm

AAAAA

Transcription

mRNA mRNA

RNA Processing

pre-mRNA

Alternative Splicing Data Sources are Large and

Growing

Microarray detectionDirect or indirect alternative splicing detection

Curated databasesSWISS-PROT and RefSeq both support annotationof experimentally supported alternative splicing

Hu et al. (2001)Genome Res 11:1237-45Yeakley et al. (2002) Nat Biotech 20:353-9

cDNA Sequencing ProjectsRIKEN sequenced >21000 full length mouse

cDNAsMany other projects underway (human, fly,

plants,…)Shinagawa et al. (2001) Nature 409:685-90

Public EST data sources (dbEST)>4.5 million human EST sequences>12 million total EST sequencesAbout 1000 new sequences per day

Boguski et al. (1993) Nat Gen 4:332-3

Gm

Nonsense-Mediated mRNA Decay

Termination codon is on the

last exon(not premature)

Mitchell and Tollervey (2001) Curr Opin Cell Biol 13:320-5

Nagy and Maquat (1998) TIBS 23:198-9Le Hir et al. (2000) Genes & Dev 14:1098-1108

Lykke-Andersen et al. (2001) Science 293:1836-9Kim et al. (2001) EMBO 20:2062-68

Ishigaki et al. (2001) Cell 106:607-17

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

Genomic DNA

pre-mRNA

mRNAExon junction complex

AAAAAAAAA

Exon Intron ExonIntron Exon

Nonsense-Mediated mRNA Decay

Termination codon > 50nt before last exon junction

(Premature Termination Codon)

Decapping and degradation

Mitchell and Tollervey (2001) Curr Opin Cell Biol 13:320-5

Nagy and Maquat (1998) TIBS 23:198-9Le Hir et al. (2000) Genes & Dev 14:1098-1108

Lykke-Andersen et al. (2001) Science 293:1836-9Kim et al. (2001) EMBO 20:2062-68

Ishigaki et al. (2001) Cell 106:607-17

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

mRNAGm

AAAAAAAAA

Interaction between EJC andrelease factors triggers NMD

Nonsense-Mediated mRNA Decay

Gm

Gm

Translated normally

Degraded by NMD

>50 nt

mRNA

mRNA

AAAAAAAAA

AAAAAAAAA

ORF

ORF

NMD is Pervasive1498 of 1500 genes surveyed

from fungi, plants, insects and vertebrates obey the PTC

ruleNagy and Maquat (1998) TIBS 23:198-9

“NMD is a critical process in normal cellular developement”

Wagner and Lykke-Andersen (2002) J Cell Sci 115:3033-8

Wang et al. (2002) J Biol Chem 277:18489-93

Renders recessive many otherwise dominant

mutationsCali and Anderson (1998) Mol Gen Genet 260:176-84

V(D)J recombination

4.3% of reviewed RefSeqs have PTCs 34% have start codon after first exon

TranscriptionalRegulation

RUST

Gene locus

pre-mRNA

productivemRNA

Protein

transcription

productivesplicing

translation

RUSTTranscriptional

RegulationGene locus

productivesplicing

Gene locus

pre-mRNA

transcription

pre-mRNA

ProductivemRNA

ProductivemRNA

Alternative Splicing Can Yield Isoforms Differentially Subjected to NMDNucleus

Prematuretermination

codon

NMD

pre-mRNA

DNA

mRNAmRNA

DNA

pre-mRNA

Nucleus

SC35 Auto-regulationSC35 Locus

SC35 pre-mRNA

Productive SC35 mRNA

SC35 protein

splicing

transcription

translation

alternativesplicing

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

Gm

Gm

SC35 Auto-regulationAlternative splicing coupled with

nonsense-mediated decay

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

SC35SC35

SC35SC35

SC35 pre-mRNA

SC35 mRNA

SC35protein

SC35 pre-mRNA

SC35 mRNA(with premature

termination codon)

SC35 Locus

SC35 pre-mRNA

Productive SC35 mRNA

SC35 protein

AAAAA

AAAAA

ORF

SC35

EST-inferred human isoforms

0 2000 4000 6000 8000

NMDCandidates

Alternative isoforms

All isoforms, including canonical

1989 (35 % of 5693)

5693

8820

10000

Canonical Splice FormsGenomic Contigs

Coding Refseqs

Association via LocusLink

Refseq mRNAs

Extract coding regions

align w/ Spidey

Refseq-Contig Pairs

≥98% id, no gaps

Construct genes from aligned Refseq exons

& intervening genomic introns (overlapchoose mRNA w/ largest number of

exons)

Refseq-coding genes

Lander et al. (2001) Nature 409: 860-921

Wheelan et al. (2001) Gen Res 11:1952-7

Pruitt, K.D. et al (2001) NAR 29: 137-40

mRNA

Exon 1 Exon 3Exon 2 Exon 4

Refseq-coding gene

Genomic DNA Sequence

Cluster ESTs w/ WU-BLAST2

≥92% id, allow gaps

Refseq-coding genes

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

Gish,(2002)(Wash.Univ.)

Align ESTs w/ sim4

Alternative Isoforms of Refseq-coding genes

Kan, et al. (2001) Gen Res 11, 889-900.

Florea, et al.,(1998)Gen Res 8, 967-74.

Identification of Alternative Isoforms

ESTs from dbEST

Use TAP to infer alternative

mRNAs

>92% identity, gaps allowed

Aligned EST 5’ end does not indicate reading frame

Class Experimental Evidence Among Our Results

Splicing Factors

AUF1, SC35SRP20, SRP30b (in C. elegans)

Sureau et al. (2001) EMBO J 20:1785-96Wilson et al. (1999) Mol Cell Bio 19:4056-64

Morrison et al. (1997) PNAS 94:9782-9785

AUF1, *10 new

Ribosomal Proteins

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

L30, S14B (in S. cerevisiae) Mitrovich & Anderson (2000) GenesDev 14:2173-84

L3, L7a, L10a, L12, *11 new

Previous and new RUST targets

Alternative Splicing

Recruitment of Sequence.

Deletion of Sequence.

*Frameshift and Truncation.

not integer # codons

Premature Stop Codons

EST LimitationsSingle pass

sequencing errors

Incompletelyprocessed transcripts

3’ end bias

Librarycontamination

Thanaraj (1999) NAR 27:2627-37

Alternative Splicing EST Analysis

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

0

500

1000

1500

2000

Did not affect

reading frame

Inserted stop

codon

Changed

reading frame

Num

ber

of EST

splice

form

s

Alternative Isoform Inference from Splice Pairs

Alternative Splice Pairs, by Mode

Alternative Splice Pairs, by Mode

Splice Pairs Generating Premature Stops

For 76% of isoforms with premature stops:

ESTs cover a PTC & splice junction downstream

EST coverage and premature stops

In 80% of these isoforms, there is a PTC in every reading frame:

Alternative polyadenlyation signals are biased against recovery

RefSeq mRNAAlternatively spliced EST, reading frame 0

Alternatively spliced EST, reading frame 2

Alternatively spliced EST, reading frame 1