Transcript Characterizing Alternative Splicing With Respect To

Characterizing Alternative
Splicing With Respect To
Protein Domains
BME 220 Project
Charlie Vaske
Overview
Background
 Methods and Data
 Preliminary Results
 Preliminary Conclusions

Big Picture
Genome
DNA
Transcriptome
RNA
Proteome
Protein
Splicing in Higher Eukaryotes
Genome:
5’ Intergenic
Gene
Intergenic
3’
Exon
3’
Transcription of pre-mRNA
5’ Exon
Intron
GT
Donor Site
Exon
AG
Acceptor Site
U1
Intron
GT
AG
U2
Splicing to mRNA
Transcriptome:
5’ Exon
Exon
Exon
3’
Alternative Splicing
5’ Exon
Intron
GT
5’
Exon
GT
GT
Intron
Exon
GT
5’ Exon
5’
pre-mRNA
Exon
AG
AG
Intron
GT
AG
Intron
GT
AG
Intron
GT
AG
Exon
GT
Intron
AG
GT
GT
GT
Exon
3’
Exon
3’
Exon
3’
Exon
3’
AG
AG
Microarrays For Alt. Splicing
Use short oligonucleotides
 Get a guess at the rate of expression of
the oligo

Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Affymetrix
Microarrays For Alt. Splicing
Exon 1
Exon 2
Exon 4
Exon 5
Exon 3
Isoform 1:
Exon 1
Exon 2
Exon 4
Exon 5
Isoform 2:
Exon 1
Exon 3
Exon 5
Probe types
Constitutive
Junction
Exon
Unique (“Cassette”)
Expression
Ideal Microarray Readings
a
Isoform 1:
b
a
Exon 1
c
Probe
d
c
Exon 2
Exon 4
Exon 5
b
Isoform 2:
a
Exon 1
e
d
Exon 3
Exon 5
e
Probe types
Constitutive
Exon
Junction
Unique (“Cassette”)
Motivation
Why alternatively splice?
 How does it affect the resulting proteins?
 Look at domains:

High level summary of protein
 ~80% of eukaryotic proteins are multi-domain
 Domains are big relative to an exon

Some Previous Work

Signatures of domain shuffling in the
human genome. Kaessmann, 2002.
Intron phase symmetry around domain
boundaries

The Effects of Alternative Splicing On
Transmembrane Proteins in the Mouse
Genome. Cline, 2004.
Half of TM proteins studied affected by altsplicing.
Method
Predict Alternative Splicing
 Predict Protein Domains
 Look for effects of Alt-Splicing on
predicted domains

“Swapping”
 “Knockout”
 “Clipping”

Microarray Design
Genes based on mRNA and EST data in
mouse
 Mapped to Feb. 2002 mouse genome
freeze
 ~500,000 probes (~66,000 sets)
 ~100,000 transcripts
 ~13,000 gene models

Technical work
Genome Space
Provided data
gene models
transcripts
Overlap
Probe to transcript mapping
Generated Data
Overlap
E@NM_021320
cc-chr10-000017.82.0
G6836022@J911445
cc-chr10-000017.91.1
G6807921@J911524_RC cc-chr10-000018.4.0
probes
Predicting Alternative Splicing
Using mouse alt-splicing microarrays
 Data from Manny Ares

8 tissues
 3 replicates of each tissue

Predicting Alternative Splicing

General Approach: Clustering, then AntiClustering
107 Clusters
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Detail View
Predicting Alternative Splicing

Cluster pairs have both anti-correlation and overlap
First Attempt at Predictions
Concerned with prediction quality
 Only took clusters-pairs with anticorrelation less than -0.9

QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
First Attempt at Predictions
Greater than -0.9 anti-correlation
 121 genes
 ~60 named genes
 Many of these have documented isoforms

Predicting Protein Domains
Used local install of InterPro
 Only used pfam

Based on sequence motifs

Liberal e-value cut-off: ~1e-10
Technical work
Genome
Space
Transcriptome
Space
Proteome
Space
mRNA
Amino Acid
Sequences
transcripts
Predicted
Domains
Predicted
Domains
Predicted
Domains
“Swapping”
I define to be:

Genome base pair annotated with >1 domain
Exon
Exon
Exon

Exon
2 isoforms share a domain, then each has a
domain of different types on the same side
Exon
Exon
Exon
Exon
“Knockout”

Cassette exon indels a predicted domain
Exon
Exon
Exon
Exon
Exon
or
Exon
Exon
Exon
Exon
Exon
“Clipping”

Lengthening/shortening of a domain
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Results: Selected Prediction

3’
C
Rabggtb
5’
N
Preliminary Conclusions
Only a few genes examined
 Analysis pipeline in infancy
 Not thoroughly tested

I do have alternative splicing events
 Example and literature suggest that some
effects will be found
