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Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains
Published on: December 13, 2014
Nonsense-mediated mRNA decay factors cure most [PSI+] prion variants
1Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Abstract:
The yeast prion [PSI+] is a self-propagating amyloid of Sup35p with a folded in-register parallel β-sheet architecture. In a genetic screen for antiprion genes, using the yeast knockout collection, UPF1/NAM7 and UPF3, encoding nonsense-mediated mRNA decay (NMD) factors, were frequently detected. Almost all [PSI+] variants arising in the absence of Upf proteins were eliminated by restored normal levels of these proteins, and [PSI+] arises more frequently in upf mutants. Upf1p, complexed with Upf2p and Upf3p, is a multifunctional protein with helicase, ATP-binding, and RNA-binding activities promoting efficient translation termination and degradation of mRNAs with premature nonsense codons. We find that the curing ability of Upf proteins is uncorrelated with these previously reported functions but does depend on their interaction with Sup35p and formation of the Upf1p-Upf2p-Upf3p complex (i.e., the Upf complex). Indeed, Sup35p amyloid formation in vitro is inhibited by substoichiometric Upf1p. Inhibition of [PSI+] prion generation and propagation by Upf proteins may be due to the monomeric Upf proteins and the Upf complex competing with Sup35p amyloid fibers for available Sup35p monomers. Alternatively, the association of the Upf complex with amyloid filaments may block the addition of new monomers. Our results suggest that maintenance of normal protein-protein interactions prevents prion formation and can even reverse the process.
Insights
Nonsense-mediated mRNA decay (NMD) factors, Upf1p and Upf3p, prevent yeast prion [PSI+] formation and propagation. Restoring these proteins cures existing prions, suggesting protein interactions inhibit amyloidogenesis.
Area of Science:
- Molecular Biology
- Protein Folding
- Yeast Genetics
Background:
- The yeast prion [PSI+] is a self-propagating amyloid aggregate of the Sup35 protein.
- Nonsense-mediated mRNA decay (NMD) pathway factors, UPF1 and UPF3, were identified in a screen for antiprion genes.
- Prion formation and propagation are influenced by cellular protein homeostasis mechanisms.
Purpose of the Study:
- To investigate the role of nonsense-mediated mRNA decay (NMD) factors in the generation and maintenance of the yeast prion [PSI+].
- To determine the mechanism by which NMD factors inhibit prion formation and propagation.
- To explore the potential of protein-protein interactions in preventing or reversing prion formation.
Main Methods:
- Genetic screening using the yeast knockout collection to identify antiprion genes.
- Assessing [PSI+] prion variants in yeast mutants lacking NMD factors (upf mutants).
- In vitro studies of Sup35p amyloid formation in the presence of Upf1p.
- Investigating the interaction between NMD factors and Sup35p.
Main Results:
- UPF1 and UPF3 were frequently detected as antiprion genes; [PSI+] arose more frequently in upf mutants.
- Restoring Upf proteins eliminated existing [PSI+] variants, indicating a curing effect.
- The prion-curing ability of Upf proteins is independent of their known mRNA decay functions but depends on interaction with Sup35p and formation of the Upf complex.
- Upf1p inhibited Sup35p amyloid formation in vitro, suggesting competition for Sup35p monomers or blockage of fiber elongation.
Conclusions:
- Nonsense-mediated mRNA decay factors (Upf proteins) play a critical role in preventing yeast prion [PSI+] formation and propagation.
- The mechanism involves direct interaction with Sup35p, potentially by competing for monomers or blocking fibril growth, rather than canonical NMD functions.
- Maintaining normal protein-protein interactions, facilitated by Upf proteins, is crucial for preventing and potentially reversing prion formation.
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