lecture 10. Genetic Interactionslecture 10. Genetic Interactions

synthetic lethalitysynthetic lethalityhigh copy suppression suppression and enhancementhigh copy suppression suppression and enhancementmechanisms of suppressionmechanisms of suppression

anc4anc4

anc1anc1

anc3anc3

anc2anc2

act1-1act1-1

act1-4act1-4

tpm1tpm1

sac6sac6

abp1abp1

non-complementationnon-complementation

synthetic lethalsynthetic lethal

synthetic slow growthsynthetic slow growth

actinactin actin actin non-non-complementercomplementer

actinactin-binding-bindingproteinprotein

Welch etWelch et alia alia Genetics 1993Genetics 1993

Anc1=mediatorAnc1=mediator

Synthetic PhenotypesSynthetic Phenotypes

GenotypeGenotype PhenotypePhenotypeyfg-1yfg-1 yfg-2yfg-2+/++/+ +/+ +/+ wild-typewild-type+/-+/- +/+ +/+ wild-typewild-type-/--/- +/+ +/+ wild-typewild-type+/++/+ -/- -/- wild-typewild-type+/++/+ +/- +/- wild-typewild-type -/--/- -/- -/- mutant mutant

Ferguson and Horvitz (1989)Ferguson and Horvitz (1989)syntheticsynthetic multivulva genesmultivulva genes

““CB1322CB1322”” = lin-8 A = lin-8 A lin-9 Blin-9 B

lin-15Alin-15A lin-15Blin-15B

Other, unrelated syntheticOther, unrelated synthetic multivulva locimultivulva locisli-1 (sli-1 (CblCbl))unc-101 (unc-101 (clathrin clathrin adaptoradaptor mu-1)mu-1)gap-1 (gap-1 (rasGAPrasGAP))

Yeast Secretory Pathway: Mutant Classes

From Alberts (ncbi.nlm)

secretion in yeastsecretion in yeast

No. Synthetic LetNo. Synthetic Let Intxns Intxns

StepStep FunctionFunction No. GenesNo. Genes SameSame Difft Difft Not SecretionNot Secretion

11 Trnslocn Trnslocn to to GolgiGolgi 2323 66 00 44

22 MaturMatur’’n n in ERin ER 1616 1010 00 22

33 Vesicle buddingVesicle budding 1212 1111 33 22

44 Vesicle fusionVesicle fusion 1818 1111 1313 11

55 Transport toTransport to Golgi Golgi 1111 22 99 33

66 Fusion with Pl MBFusion with Pl MB 3030 4343 1313 1212

77 RetrievalRetrieval 2020 1515 99 11

88 Vacuolar targetingVacuolar targeting 1616 66 66 44

99 EndocytosisEndocytosis 2020 1212 1212 2222

1010 Endosome Endosome to Vacuoleto Vacuole 77 00 33 22

1111 TotalTotal 173173 116116 6868 5353

secretion in yeastsecretion in yeast

High copy suppressionHigh copy suppression

GenotypeGenotype PhenotypePhenotypeyfg1yfg1 DNADNA+/++/+ nonenone wild-typewild-type+/-+/- nonenone wild-typewild-type-/--/- nonenone mutant mutant-/--/- yfg2(+) yfg2(+) wild-typewild-type+/++/+ yfg2(+) yfg2(+) ??

High copy suppression: yeast alpha factorHigh copy suppression: yeast alpha factor

GenotypeGenotype MatingMatingMATMAT otherother w/aw/a w/w/αα BarBar ααFFMATMATaa - - ++ ++ --MATMATαα ++ -- -- ++matmatαα11 -- -- -- -- matmatαα2 2 -- <1%<1% + + --matmatαα1 1 αα2 2 -- ++ ++ --matmatαα2 2 bar1-1bar1-1 -- ?? + + ++matmatαα2 2 ⇑⇑MFMFαα11 -- ?? + + ++

Suppressor screens, examplesSuppressor screens, examples

Suppressor ofSuppressor of Multivulva in Multivulva in C.C. eleganselegans

Activated Go-alpha in Activated Go-alpha in C.C. eleganselegans

Enhancer screens, examplesEnhancer screens, examples

Rough eye in Rough eye in DrosophilaDrosophila

pro

RING +SH2

SH3

LET-23 EGFR

SEM-5Grb2

LET-341SOS

LET-60RAS

Vulval differentiation

ARK-1

KINASE

SH2

SH3

pro

GNEF

pro

KINASE

SLI-1Cbl

Y~P

Y~P

rasGAP

Y~P

Ack-related kinase

KINASE

KINASE

KINASE

LIN-45RAF

MEK-2

MPK-1

C. C. eleganselegans

SH3

Sevenless RTK

DrkGrb2

SOSGNEF

RAS1

R7

KINASE

SH2

SH3

pro

GNEF

Y~P

Y~P

GAP

Y~P

KINASE

KINASE

KINASE

MAP-KKK

MAP-KK

MAP K

Drosophila Photoreceptor DevelopmentDrosophila Photoreceptor Development

R8 induces R7R8 induces R7

Multiple Multiple OmmatidaOmmatidain each eye:in each eye:a population assaya population assay

The fly eye consists of approximately 800 20-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 theR8 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 specifyR7'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 arelikely 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 atemperature-sensitive phenotype. A fly carrying one copy of this transgene is wildtype at 22.7oC (R7 is present).However, at 24.3oC R7 is absent

An enhancer screen for essential genes required for R7 development

sev/Y; +/+; +/+male sev/sev; +/+; P[sev-ts]/balancerX

sev/sev; */+; P[sev-ts]/+ sev/Y; */+; P[sev-ts]/+

Screen for absence of R7 in individual flies.Isolate these chromosomes by balancing.

sev/sev; +/+; P[sev-ts]/Y at 22.7oC

sev/sev; +/+; P[sev-ts]/Y at 24.3oC

sev/sev; */+; P[sev-ts]/Y at 22.7oCLook for mutation (*) that confersdominant enhancement of sev phenotype

R7 present

R7 absent

R7 absent

SH3

Sevenless RTK

DrkGrb2

SOSGNEF

RAS1

R7

KINASE

SH2

SH3

pro

GNEF

Y~P

Y~P

GAP

Y~P

KINASE

KINASE

KINASE

MAP-KKK

MAP-KK

MAP K

αα Effector

RGS

GPCR

GDP

GTP

GDPGTP

βγ

βγ Effector

Pi

Receptor is“exchange factor”

RGS is the GTPase Activating Protein

αα Effector

RGS

GPCR

GDP

GTP

GDPGTP

βγ

βγ Effector

Piα GTPase-orRGS-

G proteins Gq and Go control movement

Genotype Phenotype

Wild type wild-typeegl-30(lf) paralyzedegl-30(gf) hyperactivegoa-1(lf) hyperactivegoa-1(gf) paralyzedegl-30(lf) goa-1(lf) paralyzed

lf, loss-of-function; gf, gain-of-function

C. C. eleganselegans

Mutations that Suppress activated Goα

syIs17 syIs17; sag-4(sy433)

Before Heat Shock

AfterHeat Shock

Jane Mendel, Yvonne Jane Mendel, Yvonne HajduHajdu-Cronin, -Cronin, Wen Wen ChenChen

Suppressors of Activated Goα (Sag)

• sag-4, 8 sag-4 encodes cyclin L homologueCLASS II wild type

CLASS I hyperactive• dgk-1/sag-1 (14 alleles) encodes diacylgycerol kinase• eat-16(sy348) (p.k.a. sag-2) encodes RGS7 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

EGL-30 Gq

EGL-8 PLCβ [IP3]

[DAG]

Synaptic transmission: movement

[PIP2] DGK-1[PA]

GOA-1 GoEGL-10 RGSEAT-16 RGS

?

UNC-13 [DAG-binding] etc.

G Protein Coupled Receptors (GPCRs)

Extragenic suppression• many mechanisms--key issue is the genetic specificity of

the suppressor

‘informational’suppression

gene-nonspecificallele-specific

direct interaction?gene-specificallele-specific

epistasis (bypasssuppression)

gene-specificallele-nonspecific

suppression by compensatorychange 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 >106 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: oC mutations suppressed by mutations in repressor thatbind more tightly to operator (Pfahl 1981, J. Mol. Biol. 147: 1-10)

• protein-protein interactions?

allele-specific suppression

• null mutants are not suppressed, so notbypass suppressor

• stabilization or altered processing of mutantgene product

suppression by formation of newprotein-protein interactions

Adams + Botstein 1989.suppressors of ts actinmutants– get sac mutants. sac6 is

fimbrin, actin-binding– sac6 mutations are

missense in actin bindingdomain, increase affinityfor mutant actin

– But the affinity for wildtype actin is also increased

ACT SAC

act SAC

act sac

ACT sac

gene non-specific, allele specific

• suppression at level of gene expression:‘informational’

1. Nonsense suppression2. Frameshift suppression3. Splicing machinery4. stabilization of unstable mRNA or protein5. suppression of transposon insertion alleles

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 ofreadthrough

frameshift suppression

• extragenic suppression of frameshifts bytwo 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…

suppression by stabilization ofmessage

• mRNAs with ‘premature’ stop codons are recognized anddegraded– 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 partiallyfunctional protein can be made

AAAA

stopAUG

mRNAs with premature stop codonsproduce truncated proteins.

Expression of these from many loci can be detrimental to the animal.Cells have mechanisms of removing aberrant mRNAs

mRNAs with premature stop codons arerecognized and destroyed by nonsense mediated

decay

AAAA

SMG factors

stopAUG

AAAAdecapping and exonucleolytic cleavage

stop

Screens for suppressors of nonsensemutations 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

Hodgkin J, Papp A, Pulak R, Ambros V, Anderson P. A new kind of informational suppression in thenematode Caenorhabditis elegans. Genetics. 1989 Oct;123(2):301-13.

Mutations in the proteins required for nonsense mediated decaysuppress nonsense mutations by allowing stabilizing mRNAs withpremature stop codons.Functional proteins are made since low levels of readthrough makesome normal protein or because expression of the truncated proteincan suppress the phenotype

AAAA

stopAUG

In the absence of SMG proteins mRNAs withpremature stop codons will persist

Expression of these from many loci can be detrimental to the animal

mRNAs with premature stop codons have a low level of readthrough,these levels may be enough to rescue the mutant phenotype

Short protein fragmentis not functional orantimorphic

suppression by stabilization ofprotein

• E. coli lon protease degrades aberrantproteins

• mutations in lon suppress thermolabilemutations in many genes (RNA polymeraseetc)