Characterizing Alternative Splicing With Respect To Protein Domains

1
Characterizing Alternative Splicing With Respect
To Protein Domains

• BME 220 Project
• Charlie Vaske

2
Overview

• Background
• Methods and Data
• Preliminary Results
• Preliminary Conclusions

3
Big Picture
Genome
Transcriptome
Proteome
DNA
RNA
Protein
4
Splicing in Higher Eukaryotes
Genome
5
3
Intergenic
Intergenic
Gene
Transcription of pre-mRNA
5
3
Exon
Intron
Exon
Exon
Intron
GT
AG
AG
GT
Donor Site
Acceptor Site
U2
U1
Splicing to mRNA
5
3
Transcriptome
Exon
Exon
Exon
5
Alternative Splicing
3
5
pre-mRNA
GT
AG
6
Microarrays For Alt. Splicing

• Use short oligonucleotides
• Get a guess at the rate of expression of the oligo

Exon 1
Exon 2
Exon 4
Exon 5
Exon 3
7
AffymetrixMicroarrays 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
8
Ideal Microarray Readings
Expression
a
b
c
d
e
Probe
Isoform 1
a
c
Exon 1
Exon 2
Exon 4
Exon 5
b
Isoform 2
a
d
Exon 1
Exon 3
Exon 5
e
9
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

10
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
  alt-splicing.

11
Method

• Predict Alternative Splicing
• Predict Protein Domains
• Look for effects of Alt-Splicing on predicted
  domains
• Swapping
• Knockout
• Clipping

12
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

13
Technical work
Genome Space
Overlap
gene models
Generated Data
transcripts
Overlap
Provided data
Overlap
Probe to transcript mapping
E_at_NM_021320 cc-chr10-000017.82.0 G6836022_at_J9
11445 cc-chr10-000017.91.1 G6807921_at_J911524_
RC cc-chr10-000018.4.0
probes
14
Predicting Alternative Splicing

• Using mouse alt-splicing microarrays
• Data from Manny Ares
• 8 tissues
• 3 replicates of each tissue

15
Predicting Alternative Splicing

• General Approach Clustering, then Anti-Clustering

107 Clusters
Detail View
16
Predicting Alternative Splicing

• Cluster pairs have both anti-correlation and
  overlap

17
First Attempt at Predictions

• Concerned with prediction quality
• Only took clusters-pairs with anti-correlation
  less than -0.9

18
First Attempt at Predictions

• Greater than -0.9 anti-correlation
• 121 genes
• 60 named genes
• Many of these have documented isoforms

19
Predicting Protein Domains

• Used local install of InterPro
• Only used pfam
• Based on sequence motifs
• Liberal e-value cut-off 1e-10

20
Technical work
Genome Space
Transcriptome Space
Proteome Space
Amino Acid Sequences
mRNA
transcripts
Predicted Domains
Predicted Domains
Predicted Domains
21
Swapping

• I define to be
• Genome base pair annotated with gt1 domain
• 2 isoforms share a domain, then each has a domain
  of different types on the same side

Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
22
Knockout

• Cassette exon indels a predicted domain

Exon
Exon
Exon
Exon
Exon
or
Exon
Exon
Exon
Exon
Exon
23
Clipping

• Lengthening/shortening of a domain

Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
24
Results Selected Prediction

• Rabggtb

3 C
5 N
25
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