Alternative Splicing

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Alternative Splicing

• As an introduction to microarrays

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Human Genome

• 90,000 Human proteins, initially assumed near
  that number of genes (initial estimates 153,000)
• The 1000 cell roundworm Caenorhabditis elegans
  has 19,500 genes, corn has 40,000 genes
• Current estimates are 25,000 or fewer genes
• Alternative splicing allows different tissue
  types to perform different function with same
  gene assortment

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Implications

• 75 of human genes are subject to alternative
  editing
• faulty gene splicing leads to cancer and
  congenital diseases.
• gene therapy can use splicing

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Application

• We talked before about apoptotis when the cell
  determines it cant be repaired
• Bcl-x is a regulator of apoptotis, is
  alternatively spliced to produce either Bcl-x(L)
  that suppresses apoptosis, or Bcl-x(S) that
  promotes it.

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Spliceosome

• Five snRNA molecules U1, U2, U3, U4, U5, U6
  combine with as many as 150 proteins to form the
  spliceosome
• It recognizes sites where introns begin and end
• Cuts introns out of pre-mRNA
• joins exons

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Spliceosome

• The 5 splice site is at the beginning of the
  intron, the 3 site is at the end
• The average human protein coding gene is 28000
  nucleotides long with 8.8 exons separated by 7.8
  introns
• exons are 120 nucleotides long while introns are
  100-100,000 nucleotides long

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Splicing errors

• familial dysautonomia results from a
  single-nucleotide mutation that causes a gene to
  be alternatively spliced in nervous system tissue
• The decrease in the IKBKAP protein leads to
  abnormal nervous system development (half die
  before 30)
• gt 15 of gene mutations that cause genetic
  diseases and cancers are caused by splicing
  errors.

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Why splicing

• Each gene generates 3 alternatively spliced mRNAs
• Why so much intron (1-2 of genome is exons)?
• Mouse and human differences are almost all
  splicing
• Half of the human genome is made up of
  transposable elements, Alus being the most
  abundant (1.4 million copies)
• They continue to multiply and insert themselves
  into the genome at the rate of one insertion per
  100 human births
• mutations in the Alu can create a 5 or 3 site
  in an intron causing it to be an exon
• This mutation doesnt impact existing exons
• It only has effect when it is alternatively
  spliced in

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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
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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
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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
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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

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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.

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Method

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

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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

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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
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Predicting Alternative Splicing

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

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Predicting Alternative Splicing

• General Approach Clustering, then Anti-Clustering

107 Clusters
Detail View
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Gene Expression Measurement

• mRNA expression represents dynamic aspects of
  cell
• mRNA expression can be measured with latest
  technology
• mRNA is isolated and labeled with fluorescent
  protein
• mRNA is hybridized to the target level of
  hybridization corresponds to light emission which
  is measured with a laser

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Gene Expression Microarrays

• The main types of gene expression microarrays
• Short oligonucleotide arrays (Affymetrix)
• cDNA or spotted arrays (Brown/Botstein).
• Long oligonucleotide arrays (Agilent Inkjet)
• Fiber-optic arrays
• ...

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Affymetrix Microarrays
Raw image
1.28cm
107 oligonucleotides, half Perfectly Match mRNA
(PM), half have one Mismatch (MM) Raw gene
expression is intensity difference PM - MM
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Microarray Potential Applications

• Biological discovery
• new and better molecular diagnostics
• new molecular targets for therapy
• finding and refining biological pathways
• Recent examples
• molecular diagnosis of leukemia, breast cancer,
  ...
• appropriate treatment for genetic signature
• potential new drug targets

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Microarray Data Analysis Types

• Gene Selection
• find genes for therapeutic targets
• avoid false positives (FDA approval ?)
• Classification (Supervised)
• identify disease
• predict outcome / select best treatment
• Clustering (Unsupervised)
• find new biological classes / refine existing
  ones
• exploration

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Microarray Data Mining Challenges

• too few records (samples), usually lt 100
• too many columns (genes), usually gt 1,000
• Too many columns likely to lead to False
  positives
• for exploration, a large set of all relevant
  genes is desired
• for diagnostics or identification of therapeutic
  targets, the smallest set of genes is needed
• model needs to be explainable to biologists

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Microarray Data Classification
Microarray chips
Images scanned by laser
Gene Value D26528_at
193 D26561_cds1_at -70 D26561_cds2_at
144 D26561_cds3_at 33 D26579_at
318 D26598_at 1764 D26599_at
1537 D26600_at 1204 D28114_at
707
Datasets
New sample
Data Mining model
Prediction ALL or AML