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Alternative Splicing

This document discusses alternative splicing and its role in generating transcript and protein diversity. It describes how alternative splicing of pre-mRNA can result in different mRNA and protein isoforms from the same gene. One consequence of alternative splicing is the potential introduction of premature termination codons, which can target mRNA transcripts for degradation by the nonsense-mediated decay (NMD) pathway. The document presents examples of how alternative splicing coupled with NMD serves as a post-transcriptional regulatory mechanism for certain genes.

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448 views25 pages

Alternative Splicing

This document discusses alternative splicing and its role in generating transcript and protein diversity. It describes how alternative splicing of pre-mRNA can result in different mRNA and protein isoforms from the same gene. One consequence of alternative splicing is the potential introduction of premature termination codons, which can target mRNA transcripts for degradation by the nonsense-mediated decay (NMD) pathway. The document presents examples of how alternative splicing coupled with NMD serves as a post-transcriptional regulatory mechanism for certain genes.

Uploaded by

tendril123
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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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

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