Investigation of RpS3 in Yeast Ribosome Biogenesis

Investigation of RpS3 in Yeast Ribosome Biogenesis and Function Lewis & Clark Isaac Holeman’s Biochemistry & Molecular Biology Thesis, 2009 www.isaacholeman.org/thesis

Ribosomes produce protein www.isaacholeman.org/thesis Background Assembly - Export - RpS3

Ribosomes are complex machines www.isaacholeman.org/thesis Background Assembly - Export - RpS3

200,000 ribosomes per cell Background Assembly - Export - RpS3 www.isaacholeman.org/thesis

32 new ribosomes per second Background Assembly - Export - RpS3 www.isaacholeman.org/thesis

Assembly begins in the nucleus Background Assembly - Export - RpS3 www.isaacholeman.org/thesis

Ribosomal RNA forms core Background Assembly - Export - RpS3 www.isaacholeman.org/thesis

Add ribosomal proteins to core Background Assembly - Export - RpS3 www.isaacholeman.org/thesis

Cleave into small and large subunit Background Assembly - Export - RpS3 www.isaacholeman.org/thesis

Nuclear export is mediated Background Assembly - Export - RpS3 www.isaacholeman.org/thesis

Nuclear export is mediated Background Assembly - Export - RpS3 www.isaacholeman.org/thesis Crm1 cargos contain an NES Nucleus Cytoplasm

Large subunit export adapter model Background Assembly - Export - RpS3 www.isaacholeman.org/thesis NMD3

Small subunit adapter hypothesis Background Assembly - Export - RpS3 www.isaacholeman.org/thesis NMD3 Ltv1?

Export adapter hypothesis predicts Background Assembly - Export - RpS3 www.isaacholeman.org/thesis

Small subunit export should depend on Crm1

Small subunit export should depend on Ltv1

Ltv1 and Crm1 should interact directly

Ltv1 should bind the small subunit

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✔ ✔ ✔ ✔ ? RpS3:Ltv1 RpS3:Yar1 Does the RpS3:Ltv1 interaction function in export? Subunit:Adapter

How is RpS3 involved in yeast ribosome biogenesis and function? My Thesis Question www.isaacholeman.org/thesis

Generate a library of rps3(-) mutants Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Error prone PCR RPS3P RPS3 GFP

Screening for rps3 mutants Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis GAL-RPS3 RPS3P RPS3 GFP RPS3P RPS3 GFP

Screening for rps3 mutants Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis GAL-RPS3 RPS3P RPS3 GFP RPS3P RPS3 GFP Gal

Screening for rps3 mutants Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis GAL-RPS3 RPS3P RPS3 GFP RPS3P RPS3 GFP Gal Glu

Identify mutants by replica printing Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis

Screening for rps3 mutants Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis GAL-RPS3 RPS3P RPS3 GFP RPS3P RPS3 GFP Gal Glu

Retransform, replica print again Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Is the assay sensitive enough to identify mutants accurately?

Serial dilution assay for slow growth Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis

Base pair alterations of 6 mutants Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis P43S E47K V48I E32K, E89K I188V P192S

In which RpS3 functions are these mutants defective? Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Assembly? Nuclear Export? Translation? P43S E47K V48I E32K, E89K I188V P192S

RpS3 is on the surface of the small ribosomal subunit Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis P43S E47K V48I E32K, E89K I188V P192S

Mutations map to clusters in different domains Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis P43S E47K V48I E32K, E89K I188V P192S

Mutations map to part of RpS3 not touching RNA or ribosomal proteins Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis P43S E47K V48I E32K, E89K I188V P192S

Mutations map to part of RpS3 not touching RNA or ribosomal proteins Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Assembly? Nuclear Export? Translation?

How do we test whether rps3 mutations affect export? Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Assembly? Nuclear Export? Translation? RPS3P RPS3 GFP

Localization of GFP Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Nuclear exclusion Nuclear retention

Examples of nuclear retention, exclusion Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Wild Type Nuclear exclusion Nuclear retention

Examples of nuclear retention, exclusion Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Wild Type Plasmid 63 Plasmid 35 Nuclear exclusion Nuclear retention

Mutants 35, 45 have more GFP in their nuclei Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis

Do mutants 35, 45 map to same cluster? Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis

Can we suppress the mutant phenotype? Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis

Can we suppress the mutant phenotype? Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Mutations can shift equilibrium towards reactants:

Can we suppress the mutant phenotype? Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Le Chatelier's principle: If you increase concentration of a reactant… Mutations can shift equilibrium towards reactants:

Dilution assay tests for suppression of mutant phenotype Experiments Identifying mutants - Mapping Mutations - Screening for role in export www.isaacholeman.org/thesis Coming soon to a laboratory near you!

Summary Conclusions www.isaacholeman.org/thesis Isolated 19 plasmids, retransformed into yeast Dilution assay identified 3 additional mutants, 6 total Location of mutations mapped to structure consistent with function in export GFP microscopy suggests 2 mutants retain RpS3 in nucleus Constructed vectors to test if Ltv1 or Yar1 over-expression suppresses mutants

Thank You . Conclusions www.isaacholeman.org/thesis

Slides I cut out to keep it under 1/2 hour.. . Conclusions www.isaacholeman.org/thesis

Synthesizing a complex machine www.isaacholeman.org/thesis Background Assembly - Export - RpS3

The Challenge of Biosynthesis www.isaacholeman.org/thesis Background Assembly - Export - RpS3

The Challenge of Biosynthesis Background Assembly - Export - RpS3 www.isaacholeman.org/thesis

How do ribosomal subunits engage the export machinery? Background Assembly - Export - RpS3 www.isaacholeman.org/thesis