Conservation At silent sites

1. Conservation At silent sites

2. D GAC GCC A R CGT CGC R Codon models Synonymous substitution Nonsynonymous substitution

3. Ks rate of synonymous substitutions Ks sequence position Naïve assumption: no selection against synonymous substitutions Selection

4. L T S I CTT ACA AGC ATC CTT ACA AGC ATC CTT ACA AGC ATC L T S I G R GGG CGT GGT CGG GGA CGA G R T A ACT GCC ACG GCT ACA GCA T A Species 1 Species 2 Species 3 Ks sequence position Synonymous purifying selection (conservation) • Protein folding • Splicing regulatory elements • mRNA structure • Overlapping genes • Codon bias

5. How should we model synonymous selection? Ks

6. Testing for synonymous selection H0: free from synonymous selection → constant Ks H1: under synonymous selection → variable Ks likelihood ratio test

7. Research objective Quantify and characterize the magnitude and role of synonymous purifying selection

8. Comparative sequence data > 20 million years S.castelli S.bayanus S.mikatae S.paradoxus S.cerevisiae 70%-90% coding DNA sequence identity

9. GATCGATTC GATCGATTA GATCGGTCC GCTCGGTCC GATAGACAT Comparative sequence data 5,135 datasets of multiple sequence alignments + phylogenies (5,182 of ~6,000 S. cerevisiae genes) ? Obtained from Wapinski et al., Nature 2007

10. Under synonymous selection 54.4% (2,794) 45.6% (2,341) Not under synonymous selection

11. position Under significant synonymous selection Under synonymous selection Ks 12.4% (640) 42% (2,154) 45.6% (2,341) Not under synonymous selection

12. Codon bias

13. Synonymous selection underlies codon bias Different organisms prefer specific codons over others that encode the same amino acid R: S. cerevisiae AGA 48% AGG 21% CGA 7% CGC 6% CGG 4% CGU 14%

14. Codon bias maintains translational efficiency Translation accuracy Translation speed

15. Codon adaptation index (CAI) quantifies codon bias Sharp and Li. Nucleic Acids Res, 1987

16. Genes under synonymous selection are codon biased

18. Synonymous selection underlies codon bias synonymous selection ~ codon bias position

19. Codon bias (synonymous selection) derives from protein structure Translation speed Translation accuracy

20. Codon bias at the protein 3D structure S. cerevisiae mitochondrial NADP(+)-dependent isocitrate dehydrogenase (PDB: 2QFY)

21. codon bias core > codon bias surface S. cerevisiae mitochondrial NADP(+)-dependent isocitrate dehydrogenase (PDB: 2QFY)

22. codon bias interface > codon bias surface S. cerevisiae mitochondrial NADP(+)-dependent isocitrate dehydrogenase (PDB: 2QFY)

23. Silent mutations may change 3D structure

26. MDR1 is a member of the ABC transporter family. They pump drugs out of the cell utilizing ATP, which change conformation of the protein. These proteins were shown to induce multi-drug resistance in various cancers.

27. C3435T is a synonymous SNP that was reported to be a risk factor for several diseases such as Parkinson’s diseases, colon cancer, and renal epithelial tumor. • It can be either because: • Change in mRNA level • Change in splicing • Linkage disequilibrium with other causative SNPs • Something else

28. Bodipy FACS analysis. In purple – cell transfected with empty vector All other colors – cell trasfected with a vector containing MDR1 (various haplotypes) MDR1 pumps the drug (Bodipy) out of the cells.

29. All other colors – cell trasfected with a vector containing MDR1 – various haplotypes The inhibitor works differently on the various haplotypes

30. Trypsin works differently on the various haplotypes

31. They showed that synonymous substitutions did not change protein levels but rather the structure. This was shown by differential response to specific antibodies. Important for linking SNPs to diseases.

32. Silent mutations in HIV-1

33. Conservation of Ks in pol Mayrose et al. Bioinformatics/ISMB (2007)

34. Conservation of Ks in pol (zoom in) DNA flap CTS ? cPPT

35. cPPT A This region serves as a primer for the reverse transcriptase in the synthesis of the plus-strand DNA. cPPT

36. CTS = Central Termination Sequence A CTS The CTS is involved in the nuclear import of the HIV-1 genome.

37. In Pol one region is of unknown function ????

38. Silent mutations -5' mRNA region

40. Kudla et al. showed that the levels of GFP – which is a protein whose gene can easily be inserted into a host genome and its levels can then be easily quantified, are strongly affected by the secondary structure of the 5’ end of the mRNA.

41. Non- stable mRNA secondary structure at the 5’ end -> higher GFP level. Stable mRNA Non stable mRNA

42. Mechanism: stable secondary structures at the 5’ end of the mRNA obstruct ribosome binding to the mRNA and result with lower protein levels

43. Based on that we hypothesized that the 5’ end of the mRNA should show signals of strong synonymous selection. This is exactly what we found in our yeast data… In addition, we found that the codon bias is reduced at this region, as to allow non-stable mRNA structures.