Transcript Evolutionary change in proteins 2

Alternative splicing and evolution
Daniel Jeffares
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Part I.
Alternative Splicing and
Evolution
Evolutionary change in proteins 1. Single amino acids.
Evolutionary change in proteins 2.
Exon conservation/extension/deletion.
Evolutionary change in proteins 3. Domain shuffling.
Alternative splicing is the process where one gene
produces more than one type of mRNA
DNA
Environment
Development
mRNA
Cell type 1
80%
20%
Cell type 2
10%
90%
Cell type 3
absent
100%
Image from Nuclear Protein Database (NPD)
Many cellular factors may affect
which splice variant is produced
Pre-mRNA
RNA binding proteins
Pre-RNA secondary structure
Small ncRNAs?
Protein:protein complexes
Other mRNAs?
mRNA
Evolution of Transcripts is
Second-Order Evolution
there are two ways the splicing of one gene can change:
mutations in cis
mutations in trans
DNA
1. The phenotype is determined by the proteome &
transcriptome.
2. Selection acts on the phenotype, and is blind to the
genotype.
Therefore: two species/individuals that have different forms
of a protein will be selected differently - even if the genes
DNA sequence is identical.
DNA
DNA
80%
20%
mRNA
mRNA
10%
Species #1 (cell type1)
90%
Species #2 (cell type1)
Implications of alternative splicing in the
evolution of a protein:
’Trying out’ a new domain
main splice site weak splice site present in
intron
weak splice site
strengthened by a
mutation
initial protein
two proteins forms produced,
one with a new domain derived
from intron sequence
both splice sites now used
intron sequences evolve fast
this is ‘free diversity’
(the old protein remains)
Part II: some previous
studies
Overall rare/common exons are similar
between mouse & human
Modrek 2003
Human-mouse data
Modrek 2003
Alt. Exons that encode
entire domains are selected for.
Kriventseva 2003
Alternative splice sites are more likely to
fall between domains than constitutive
exons.
Kriventseva 2003
Identification of ‘new’ exonic regions
by alignment.
Fyodor 2003
New exons are shorter than average
Fyodor 2003
Part III.
Detecting alternative splicing
in
Caenorhabditis nematodes
C. elegans &C. briggsae
diverged about 25-125 MYA
Both genomes are complete
They are well studied model
Systems and are very easy to
grow in the lab.
How do splice forms evolve?
How quickly?
20%
80%
50%
50%
C. elegans
100%
0%
C. briggsae
Testing Hypothesis about
Evolutionary Change
Hypothesis:
Alternative splicing has contributed to the
phenotypic and physiological diversity of
metazoans.
Expect:
Genes that are used just to maintain basic cellular properties of the cell
will evolve more slowly than ‘developmental body pattern genes’.
Testing Hypothesis about Evolutionary Change
Hypothesis: Alternative
splicing has contributed to
the phenotypic and
physiological diversity of
metazoans.
Expect: Genes that are
used just to maintain basic
cellular functions of the
cell will evolve more slowly
than ‘developmental body
pattern genes’.
Basic cellular function genes:
Conserved in all eukaryotes
 Highly expressed, constitutive
 Lethal RNAi phenotype

Developmental body pattern
genes:
Not so highly conserved across eukaryotes
 May not be highly expressed
 Expression developmentally regulated
 Altered body RNAi phenotype

Testing Hypothesis about
Evolutionary Change
Basic cellular
function genes
Changes in splicing?
Rate of change fast or slow?
Developmental
Body pattern genes
Changes in splicing?
Rate of change fast or slow?
Exon Structure Conservation
RT-PCR method
Strengths:
•simple
•Sensitive (PCR)
•Accurate (std curves)
Limitations:
•Internal changes only
(about 300 genes)
•Cant be scaled up
Microarray Method of
Splice Variant
Detection
Each spot is the signal from one probe
The colour is transformed into number by a scanner
Capture probe design
1
2A
1
3
2B
3
Fractions of "spliceforms"
0.0
A) 1000 ppm:
channel1: 01-2A-03
channel1:
01-03
channel2: 01-2A-03
channel2:
0.4
0.6
0.8
1.0
Our arrays return the
correct ratios !
01-03
B) 1000 ppm:
channel1: 01-2A-03
channel1: 01-2B-03
channel2: 01-2A-03
Artificial spliceforms
0.2
channel2: 01-2B-03
This technological
advance has not
been achieved before.
C) 1000 ppm:
channel1: 01-2A-03
channel1: 01-2B-03
channel2: 01-2A-03
channel2: 01-2B-03
D) 100 ppm:
channel1: 01-2A-03
Input RNA:
real ratio of 83:17%
channel1: 01-2B-03
channel2: 01-2A-03
channel2: 01-2B-03
E) 10 ppm:
channel1: 01-2A-03
channel1: 01-2B-03
channel2: 01-2A-03
channel2: 01-2B-03
Calculated: the numbers
the array analysis returns
Microarray Method of Splice Variant
Detection
Strengths:
• Can be scaled up (to an entire genome…)
• Any splice variant type
• Amenable to high throughput mathmatics
& stats.
Limitations:
• Not very accurate
• Complex, expensive
Part IV: summary and
genomic perspective
•Metazoans arose
About 900 MYA
Tree topology from
Glenner et al. In Press
Implications of alternative
splicing for evolution
• alternative splicing affects the way that genomes evolve and the
way that we think about genome complexity
•How many proteins are produced in eukaryotic genomes?
•How many genes do you need to make a complex multicellular
organism?
•How can the production of many splice variants contribute to the
exploration of ’genomic sequence space’?
• How stable are splice sites in evolution?
How many genes do you need to make a
complex multicellular organism?
No. cell types
Species
Genes
1
Mycoplasma genitalium (B)
470
1
Haemophilus influenzae (B)
1 709
1
Eschericia coli (B)
4 288
1
Archaeoglobus fulgidus (A)
2 436
1
Methanococcus janaschii (A)
1 738
2
Bacillus subtilis (B)
4 100
2
Caulobacter crescentus (B)
4 100
3
Saccharomyces cerevisiae (E)
6 241
~30
Arabidopsis thaliana (E)
24 000
~50*
Caenorhabditis elegans (E)
18 424
~50
Drosophila melanogaster (E)
13 601
~120*
Homo sapiens (E)
30 000
*C. elegans has 300 neurons, humans have 10 billion
Has alternative splicing allowed complexity to evolve?
Summary
• Proteins evolve by many processes over long periods of time
• Most genes in complex eukaryotes are alternatively spliced
• It is not known how quickly alternative splicing evolves
•We will compare orthologous transcripts in two species
of nematodes to examine this ‘rate of evolution’
Using microarrays and RT-PCR
Our arrays work effectively with synthetic RNAs, but are not
very sensitive
• RT-PCR is sensitive, but cant be scaled up