Alternative splicing and Alternative splicing and evolutionevolution

                                         

Daniel JeffaresDaniel Jeffares

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Part I. Part I. Alternative Splicing and Alternative Splicing and

EvolutionEvolution

Evolutionary change in proteins 1. Single amino acids.

Evolutionary change in proteins 2. Exon conservation/extension/deletion.

Evolutionary change in proteins 3. Domain shuffling.

EnvironmentDevelopment

Alternative splicing is the process where Alternative splicing is the process where one gene produces more than one type of one gene produces more than one type of

mRNAmRNA

DNA

mRNA

80% 20%Cell type 1

10% 90%Cell type 2

absent 100%Cell type 3

Image from Nuclear Protein Database (NPD)

Many cellular factors may affect Many cellular factors may affect which splice variant is producedwhich splice variant is produced

Pre-mRNA

mRNA

RNA binding proteins

Pre-RNA secondary structure

Other mRNAs?

Small ncRNAs?

Protein:protein complexes

Evolution of Transcripts is Evolution of Transcripts is Second-Order EvolutionSecond-Order Evolution

there are two ways the splicing of one gene can change:

DNA

mutations in trans

mutations in cis

80% 20%

1. The phenotype is determined by the proteome & 1. The phenotype is determined by the proteome & transcriptome.transcriptome.2. Selection acts on the phenotype, and is blind to 2. Selection acts on the phenotype, and is blind to the genotype.the genotype.

Therefore: two species/individuals that have Therefore: two species/individuals that have different forms of a protein will be selected different forms of a protein will be selected differently - even if the genes DNA sequence isdifferently - even if the genes DNA sequence is identical.identical.

DNA

mRNA

DNA

mRNA

10% 90%

Species #1 (cell type1) Species #2 (cell type1)

Implications of alternative splicing in the Implications of alternative splicing in the evolution of a protein:evolution of a protein:

’Trying out’ a new domain’Trying out’ a new domain

main splice site initial proteinweak splice site present in intron

both splice sites now used

weak splice site strengthened by a

mutationtwo proteins forms produced, one with a new domain derived from intron sequence

intron sequences evolve fast this is ‘free diversity’(the old protein remains)

Part II: some Part II: some previous studiesprevious studies

Overall rare/common exons are similar Overall rare/common exons are similar between mouse & human between mouse & human

Modrek 2003

Human-mouse dataHuman-mouse data

Modrek 2003

Alt. Exons that encode Alt. Exons that encode entire domainsentire domains are selected for. are selected for.

Kriventseva 2003

Alternative splice sites are more Alternative splice sites are more likely to fall likely to fall betweenbetween domains than domains than

constitutive exons.constitutive exons.

Kriventseva 2003

Identification of ‘new’ exonic regions Identification of ‘new’ exonic regions

by alignment.by alignment.

Fyodor 2003

New exons are shorter than New exons are shorter than averageaverage

Fyodor 2003

Part III. Part III. Detecting alternative Detecting alternative

splicing in splicing in Caenorhabditis Caenorhabditis

nematodesnematodes C. elegans &C. briggsae diverged about 25-125 MYA

Both genomes are complete

They are well studied model Systems and are very easy togrow in the lab.

How do splice forms How do splice forms evolve?evolve?

How quickly?How quickly?

20% 80% 50% 50% 100% 0%

C. elegans C. briggsae

Testing Hypothesis about Testing Hypothesis about Evolutionary ChangeEvolutionary Change

Hypothesis:Hypothesis:

Alternative splicing has contributed to Alternative splicing has contributed to the phenotypic and physiological the phenotypic and physiological

diversity of metazoans.diversity of metazoans.Expect:Expect:

Genes that are used just to maintain basic cellular properties Genes that are used just to maintain basic cellular properties of the cell will evolve more slowly than ‘developmental body of the cell will evolve more slowly than ‘developmental body

pattern genes’.pattern genes’.

Testing Hypothesis about Evolutionary Testing Hypothesis about Evolutionary ChangeChange

Basic cellular function genes:Basic cellular function genes: Conserved in all eukaryotesConserved in all eukaryotes Highly expressed, constitutiveHighly expressed, constitutive Lethal RNAi phenotypeLethal RNAi phenotype

Developmental body pattern Developmental body pattern genes:genes: Not so highly conserved across eukaryotesNot so highly conserved across eukaryotes May not be highly expressedMay not be highly expressed Expression developmentally regulatedExpression developmentally regulated Altered body RNAi phenotypeAltered body RNAi phenotype

Hypothesis: Hypothesis: Alternative Alternative splicing has contributed splicing has contributed to the phenotypic and to the phenotypic and physiological diversity of physiological diversity of metazoans.metazoans.

Expect: Genes that are Expect: Genes that are used just to maintain used just to maintain basic cellular basic cellular functionsfunctions of the cell of the cell will evolve more slowly will evolve more slowly than ‘than ‘developmental developmental body pattern genes’body pattern genes’..

Testing Hypothesis about Testing Hypothesis about Evolutionary ChangeEvolutionary Change

Basic cellular Basic cellular

function genesfunction genes

DevelopmentalDevelopmental

Body pattern genesBody pattern genes

Changes in splicing?Rate of change fast or slow?

Changes in splicing?Rate of change fast or slow?

Exon Structure Exon Structure ConservationConservation

RT-PCR methodRT-PCR methodStrengths: •simple•Sensitive (PCR)•Accurate (std curves)

Limitations: •Internal changes only (about 300 genes)•Cant be scaled up

Microarray Microarray Method of Splice Method of Splice Variant DetectionVariant Detection

Each spot is the signal from one probe

The colour is transformed into number by a scanner

Capture probe designCapture probe design

1 2A 3

1 3

2B

Quantification of spliceforms

0.0 0.2 0.4 0.6 0.8 1.0

A) 1000 ppm:

channel1: 01-2A-03

channel1: 01-03

channel2: 01-2A-03

channel2: 01-03

B) 1000 ppm:

channel1: 01-2A-03

channel1: 01-2B-03

channel2: 01-2A-03

channel2: 01-2B-03

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

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

Artificial spliceforms

Fractions of "spliceforms"

Calculated

TRUE

Input RNA: real ratio of 83:17%

Calculated: the numbers the array analysis returns

Our arrays return thecorrect ratios !

This technological advance has not

been achieved before.

Microarray Method of Splice Microarray Method of Splice Variant DetectionVariant 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 Part IV: summary and genomic and genomic perspectiveperspective

•Metazoans aroseAbout 900 MYA

Tree topology from Glenner et al. In Press

Implications of alternative Implications of alternative splicing for evolutionsplicing 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 How many genes do you need to make a complex multicellular organismcomplex multicellular organism??

No. cell typesNo. cell types SpeciesSpecies GeneGeness

11 Mycoplasma genitalium (B)Mycoplasma genitalium (B) 470470

11 Haemophilus influenzae (B)Haemophilus influenzae (B) 1 7091 709

11 Eschericia coli (B)Eschericia coli (B) 4 2884 288

11 Archaeoglobus fulgidus (A)Archaeoglobus fulgidus (A) 2 4362 436

11 Methanococcus janaschii (A)Methanococcus janaschii (A) 1 7381 738

22 Bacillus subtilis (B)Bacillus subtilis (B) 4 1004 100

22 Caulobacter crescentus (B)Caulobacter crescentus (B) 4 1004 100

33 Saccharomyces cerevisiae (E)Saccharomyces cerevisiae (E) 6 2416 241

~30~30 Arabidopsis thaliana (E)Arabidopsis thaliana (E) 24 00024 000

~50*~50* Caenorhabditis elegans (E)Caenorhabditis elegans (E) 18 18 424424

~50~50 Drosophila melanogaster (E)Drosophila melanogaster (E) 13 13 601601

~120*~120* Homo sapiens (E)Homo sapiens (E) 30 30 000000

*C. elegans has 300 neurons, humans have 10 billion Has alternative splicing allowed complexity to evolve?

SummarySummary• 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-PCROur arrays work effectively with synthetic RNAs, but are not very sensitive• RT-PCR is sensitive, but cant be scaled up