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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. Transcriptome. Proteome. DNA. RNA. Protein. Splicing in Higher Eukaryotes.

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Presentation Transcript
overview
Overview
  • Background
  • Methods and Data
  • Preliminary Results
  • Preliminary Conclusions
big picture
Big Picture

Genome

Transcriptome

Proteome

DNA

RNA

Protein

splicing in higher eukaryotes
Splicing in Higher Eukaryotes

Genome:

5’

3’

Intergenic

Gene

Intergenic

Transcription of pre-mRNA

5’

3’

Exon

Intron

Exon

Intron

Exon

GT

AG

GT

AG

Donor Site

Acceptor Site

U2

U1

Splicing to mRNA

Transcriptome:

5’

3’

Exon

Exon

Exon

alternative splicing

Exon

Intron

Exon

Intron

Exon

GT

GT

AG

AG

GT

GT

AG

AG

5’

3’

Exon

Intron

Exon

Intron

Exon

GT

GT

AG

GT

AG

5’

3’

Exon

Intron

Exon

AG

5’

3’

Exon

Intron

Exon

GT

GT

AG

GT

AG

Alternative Splicing

5’

3’

pre-mRNA

GT

AG

microarrays for alt splicing
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

affymetrix microarrays for alt splicing

Probe types

Constitutive

Junction

Exon

Unique (“Cassette”)

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

ideal microarray readings

Probe types

Constitutive

Exon

Junction

Unique (“Cassette”)

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

motivation
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
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.

method
Method
  • Predict Alternative Splicing
  • Predict Protein Domains
  • Look for effects of Alt-Splicing on predicted domains
    • “Swapping”
    • “Knockout”
    • “Clipping”
microarray design
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
Technical work

Genome Space

Overlap

gene models

Generated Data

transcripts

Overlap

Provided data

Overlap

Probe to transcript mapping

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
Predicting Alternative Splicing
  • Using mouse alt-splicing microarrays
  • Data from Manny Ares
    • 8 tissues
    • 3 replicates of each tissue
predicting alternative splicing15
Predicting Alternative Splicing
  • General Approach: Clustering, then Anti-Clustering

107 Clusters

Detail View

predicting alternative splicing16
Predicting Alternative Splicing
  • Cluster pairs have both anti-correlation and overlap
first attempt at predictions
First Attempt at Predictions
  • Concerned with prediction quality
  • Only took clusters-pairs with anti-correlation less than -0.9
first attempt at predictions18
First Attempt at Predictions
  • Greater than -0.9 anti-correlation
  • 121 genes
  • ~60 named genes
  • Many of these have documented isoforms
predicting protein domains
Predicting Protein Domains
  • Used local install of InterPro
  • Only used pfam

Based on sequence motifs

  • Liberal e-value cut-off: ~1e-10
technical work20
Technical work

Genome Space

Transcriptome Space

Proteome Space

Amino Acid Sequences

mRNA

transcripts

Predicted Domains

Predicted Domains

Predicted Domains

swapping
“Swapping”

I define to be:

  • Genome base pair annotated with >1 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

knockout
“Knockout”
  • Cassette exon indels a predicted domain

Exon

Exon

Exon

Exon

Exon

or

Exon

Exon

Exon

Exon

Exon

clipping
“Clipping”
  • Lengthening/shortening of a domain

Exon

Exon

Exon

Exon

Exon

Exon

Exon

Exon

Exon

preliminary conclusions
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