lecture 10. Genetic Interactions synthetic lethality high copy suppression suppression and enhancement mechanisms of suppression

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1 lecture 10. Genetic Interactions synthetic lethality high copy suppression suppression and enhancement mechanisms of suppression

2 actin act1-1 act1-4 actin non- complementer anc4 anc1 anc3 anc2 actin-binding protein tpm1 sac6 abp1 Anc1=mediator Welch et alia Genetics 1993 non-complementation synthetic lethal synthetic slow growth

3 Synthetic Phenotypes Genotype Phenotype yfg-1 yfg-2 +/+ +/+ wild-type +/- +/+ wild-type -/- +/+ wild-type +/+ -/- wild-type +/+ +/- wild-type -/- -/- mutant

4 Ferguson and Horvitz (1989) synthetic multivulva genes CB1322 = lin-8 A lin-9 B lin-15a lin-15b Other, unrelated synthetic multivulva loci sli-1 (Cbl( Cbl) unc-101 (clathrin( adaptor mu-1) gap-1 (rasgap( rasgap)

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8 Yeast Secretory Pathway: Mutant Classes From Alberts (ncbi.nlm)

9 secretion in yeast

10 Step Function No. Genes No. Synthetic Let Intxns Same Difft 1 Trnslocn to Golgi Matur n in ER Vesicle budding Vesicle fusion Transport to Golgi Fusion with Pl MB Retrieval Vacuolar targeting Endocytosis Endosome to Vacuole Total Not Secretion

11 secretion in yeast

12 High copy suppression Genotype Phenotype yfg1 DNA +/+ none +/- none wild-type wild-type -/- none mutant -/- yfg2(+) wild-type +/+ yfg2(+)?

13 High copy suppression: yeast alpha factor Genotype Mating MAT other w/a w/α Bar αf MATa MATα matα matα <1% matα1 α matα2 matα2 bar ? MFα ?

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16 Suppressor screens, examples Suppressor of Multivulva in C. elegans Activated Go-alpha in C. elegans Enhancer screens, examples Rough eye in Drosophila

17 C. elegans LET-23 EGFR SLI-1 Cbl SH2 RING + pro KINASE SEM-5 Grb2 Y~P SH3 SH2 pro Y~P SH3 pro Y~P KINASE rasgap GNEF LET-60 RAS LET-341 SOS KINASE KINASE KINASE LIN-45 RAF MEK-2 MPK-1 ARK-1 Ack-related kinase Vulval differentiation

18 Sevenless RTK KINASE Drk Grb2 Y~P SH3 SH2 Y~P SH3 Y~P pro GAP GNEF RAS1 SOS GNEF KINASE KINASE KINASE MAP- KKK MAP- KK MAP K R7

19 Drosophila Photoreceptor Development R8 induces R7

20 Multiple Ommatida in each eye: a population assay

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22 An enhancer screen for essential genes required for R7 development The fly eye consists of approximately cell repeating units known as ommatidia. Each ommatidium consists of eight photoreceptor neurons (R1-R8), four lens secreting cone cells and eight additional accessory cells. The ommatidia arise from an undifferentiated epithelium by a series of cell interactions. We will only consider an interaction between the R8 and presumptive R7 cells that determines the fate of R7. The R7 photoreceptor detects light in the UV range. Screens for mutants with ommatidia that lack R7 cells identified three genes: sevenless (sev), bride of sevenless (Boss) and seven-in-abstentia (sina). Adult flies homozygous for mutations in any of these genes have ommatidia that lack an R7 cell and contain an additional cone cell. In the absence of R7 differentiation, the presumptive R7 cell becomes a cone cell. sev and sina are a receptor tyrosine kinase and a nuclear protein, respectively, and both genes act in R7 to specify R7's fate. boss appears to encode the ligand for the Sev receptor tyrosine kinase, and in contrast to sev and sina, acts in R8 cell to specify R7's fate. Now consider the problem that many genes functioning downstream of receptor tyrosine kinse receptor activation are likely to be required for other tyrosine kinase signaling pathways that are required for the viability of the organism. How can one use the fly eye to identify such mutations in such genes. Make a partially active mutant version of sev and introduce it into a sev mutant background. These flies have a temperature-sensitive phenotype. A fly carrying one copy of this transgene is wildtype at 22.7 o C (R7 is present). However, at 24.3 o C R7 is absent

23 sev/y; +/+; +/+male X sev/sev; +/+; P[sev-ts]/balancer sev/sev; */+; P[sev-ts]/+ sev/y; */+; P[sev-ts]/+ Screen for absence of R7 in individual flies. Isolate these chromosomes by balancing. R7 present sev/sev; +/+; P[sev-ts]/Y at 22.7 o C R7 absent sev/sev; +/+; P[sev-ts]/Y at 24.3 o C R7 absent sev/sev; */+; P[sev-ts]/Y at 22.7 o C Look for mutation (*) that confers dominant enhancement of sev phenotype

24 Sevenless RTK KINASE Drk Grb2 Y~P SH3 SH2 Y~P SH3 Y~P pro GAP GNEF RAS1 SOS GNEF KINASE KINASE KINASE MAP- KKK MAP- KK MAP K R7

25 Receptor is exchange factor GPCR γ β Effector GTP GDP GDP α γ β GTP α Effector Pi RGS RGS is the GTPase Activating Protein

26 GPCR γ β Effector GTP GDP GDP α γ β GTP α Effector Pi RGS α GTPaseor RGS-

27 G proteins Gq and Go control movement C. elegans Genotype Wild type egl-30(lf) egl-30(gf) goa-1(lf) goa-1(gf) egl-30(lf) goa-1(lf) Phenotype wild-type paralyzed hyperactive hyperactive paralyzed paralyzed lf, loss-of-function; gf, gain-of-function

28 Mutations that Suppress activated Goα syis17 syis17; sag-4(sy433) Before Heat Shock After Heat Shock Jane Mendel, Yvonne Hajdu-Cronin, Hajdu-Cronin, Wen Chen

29 Suppressors of Activated Goα (Sag) CLASS I hyperactive dgk-1/sag-1 (14 alleles) encodes diacylgycerol kinase eat-16(sy348) (p.k.a. sag-2) encodes RGS7 homologue CLASS II wild type sag-4, 8 sag-4 encodes cyclin L homologue CLASS III Egg-laying defective sag-3, 5 sag-3 encodes Heat Shock Factor CLASS IV wild type sag-6 CLASS V Egg-laying defective sag-7 Yvonne Hajdu-Cronin & Wen Chen

30 EGL-30 Gq G Protein Coupled Receptors (GPCRs) EAT-16 RGS? EGL-10 RGS GOA-1 Go EGL-8 PLCβ [IP3] [PIP2] [DAG] DGK-1 [PA] UNC-13 [DAG-binding] etc. Synaptic transmission: movement

31 Extragenic suppression many mechanisms--key issue is the genetic specificity of the suppressor gene-specific allele-nonspecific gene-specific allele-specific gene-nonspecific allele-specific epistasis (bypass suppression) direct interaction? informational suppression

32 suppression by compensatory change in direct interactor? Lock and Key model: binding site is restored in general a very rare event as target size is 1(or a few) bp- -need screens of >10 6 genomes RNA-RNA interactions: restoration of base pairing (nonsense suppression) splice site suppression e.g. Lesser + Guthrie 1993 Science 262: 1982 protein-dna interactions lac operon: o C mutations suppressed by mutations in repressor that bind more tightly to operator (Pfahl 1981, J. Mol. Biol. 147: 1-10) protein-protein interactions?

33 allele-specific suppression null mutants are not suppressed, so not bypass suppressor stabilization or altered processing of mutant gene product

34 suppression by formation of new protein-protein interactions Adams + Botstein suppressors of ts actin mutants get sac mutants. sac6 is fimbrin, actin-binding sac6 mutations are missense in actin binding domain, increase affinity for mutant actin But the affinity for wild type actin is also increased ACT act act ACT SAC SAC sac sac

35 gene non-specific, allele specific suppression at level of gene expression: informational 1. Nonsense suppression 2. Frameshift suppression 3. Splicing machinery 4. stabilization of unstable mrna or protein 5. suppression of transposon insertion alleles

36 nonsense suppression conditional amber mutations in many T4 genes (Epstein et al) grow on one E coli strain (CR63) but not on B cause premature termination suppression due to mutant trna that can recognize amber codon UAG and insert amino acid (usually Trp; codon is UGG) amber suppressor strains are a bit sick because of readthrough

37 frameshift suppression extragenic suppression of frameshifts by two mechanisms limitation of Trp-tRNA other trnas loosely bind to codon (mismatch) and allow frameshifting also mutant trna with 4-base anticodon now reads frameshift as a 3-base codon

38 suppression by stabilization of message mrnas with premature stop codons are recognized and degraded nonsense mediated decay/ mrna surveillance Upf pathway (yeast), SMG pathway (worms) get rid of aberrant mrnas before they get to ribosome some nonsense mutations can be suppressed if partially functional protein can be made

39 mrnas with premature stop codons produce truncated proteins. AUG stop AAAA Expression of these from many loci can be detrimental to the animal. Cells have mechanisms of removing aberrant mrnas

40 mrnas with premature stop codons are recognized and destroyed by nonsense mediated decay AUG stop AAAA SMG factors stop AAAA decapping and exonucleolytic cleavage

41 Screens for suppressors of nonsense mutations revealed smg genes smg-1 phosphatidylinositol-3 kinase homolog smg-2 Upf1 helicase homolog, phosphoprotein smg-3 Upf2 homolog smg-4 Upf3 homolog smg-5 novel, binds SMG-7 smg-6 -- smg-7 novel, binds SMG-5 Mutations in the proteins required for nonsense mediated decay suppress nonsense mutations by allowing stabilizing mrnas with premature stop codons. Functional proteins are made since low levels of readthrough make some normal protein or because expression of the truncated protein can suppress the phenotype Hodgkin J, Papp A, Pulak R, Ambros V, Anderson P. A new kind of informational suppression in the nematode Caenorhabditis elegans. Genetics Oct;123(2):

42 In the absence of SMG proteins mrnas with premature stop codons will persist Expression of these from many loci can be detrimental to the animal AUG stop AAAA Short protein fragment is not functional or antimorphic mrnas with premature stop codons have a low level of readthrough, these levels may be enough to rescue the mutant phenotype

43 suppression by stabilization of protein E. coli lon protease degrades aberrant proteins mutations in lon suppress thermolabile mutations in many genes (RNA polymerase etc)