1. Alternative splicing and evolution Daniel Jeffares

2. Part I. Alternative Splicing and Evolution

3. Evolutionary change in proteins 1. Single amino acids.

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

5. Evolutionary change in proteins 3. Domain shuffling.

6. Environment Development Alternative splicing is the process where one gene produces more than one type of mRNA DNA mRNA 80%20%Cell type 1 10%90%Cell type 2 absent100%Cell type 3

7. Image from Nuclear Protein Database (NPD)

8. Many cellular factors may affect which splice variant is produced Pre-mRNA mRNA RNA binding proteins Pre-RNA secondary structure Other mRNAs? Small ncRNAs? Protein:protein complexes

9. Evolution of Transcripts is Second-Order Evolution there are two ways the splicing of one gene can change: DNA mutations in trans mutations in cis

10. 80% 20% 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 mRNA DNA mRNA 10% 90% Species #1 (cell type1)Species #2 (cell type1)

11. Implications of alternative splicing in the evolution of a protein: ’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 mutation two proteins forms produced, one with a new domain derived from intron sequence intron sequences evolve fastthis is ‘free diversity’ (the old protein remains)

12. Part II: some previous studies

13. Overall rare/common exons are similar between mouse & human Modrek 2003

14. Human-mouse data Modrek 2003

15. Alt. Exons that encode entire domains are selected for. Kriventseva 2003

16. Alternative splice sites are more likely to fall between domains than constitutive exons. Kriventseva 2003

17. Identification of ‘new’ exonic regions by alignment. Fyodor 2003

18. New exons are shorter than average Fyodor 2003

19. Part III. Detecting alternative splicing in Caenorhabditis nematodes 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.

20. How do splice forms evolve? How quickly? 20%80%50% 100%0% C. elegansC. briggsae

21. 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’.

22. Testing Hypothesis about Evolutionary Change Basic cellular function genes: Conserved in all eukaryotes Conserved in all eukaryotes Highly expressed, constitutive Highly expressed, constitutive Lethal RNAi phenotype Lethal RNAi phenotype Developmental body pattern genes: Not so highly conserved across eukaryotes Not so highly conserved across eukaryotes May not be highly expressed May not be highly expressed Expression developmentally regulated Expression developmentally regulated Altered body RNAi phenotype Altered body RNAi phenotype 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’.

23. Testing Hypothesis about Evolutionary Change Basic cellular function genes Developmental Body pattern genes Changes in splicing? Rate of change fast or slow? Changes in splicing? Rate of change fast or slow?

24. Exon Structure Conservation

25. RT-PCR method Strengths: simple Sensitive (PCR) Accurate (std curves) Limitations: Internal changes only (about 300 genes) Cant be scaled up

26. Microarray Method of Splice Variant Detection

28. Each spot is the signal from one probe The colour is transformed into number by a scanner

29. Capture probe design 12A3 13 2B

30. Input RNA: real ratio of 83:17% Calculated: the numbers the array analysis returns Our arrays return the correct ratios ! This technological advance has not been achieved before.

31. 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

32. Part IV: summary and genomic perspective

33. Metazoans arose About 900 MYA Tree topology from Glenner et al. In Press

34. 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?

35. How many genes do you need to make a complex multicellular organism? No. cell types SpeciesGenes 1 Mycoplasma genitalium (B) 470 470 1 Haemophilus influenzae (B) 1 709 1 Eschericia coli (B) 4 288 1 Archaeoglobus fulgidus (A) 2 436 1 Methanococcus janaschii (A) 1 738 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?

36. 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