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Investigating the combined burden of transcription and quality control errors in yeast

Klucevsek, Kristin (2012) Investigating the combined burden of transcription and quality control errors in yeast. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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The Rtf1 subunit of the Paf1 complex is required for specific conserved transcription-coupled histone modifications, including histone H2B lysine 123 monoubiquitylation. In Saccharomyces cerevisiae, deletion of RTF1 is lethal in the absence of Rkr1, a ubiquitin-protein ligase involved in the destruction of nonstop proteins, which arise from mRNAs lacking stop codons or translational read-through into the poly(A) tail. To understand the combined requirement for H2B ubiquitylation and protein quality control in yeast, I performed a transposon-based mutagenesis screen to identify suppressors of rtf1Δ rkr1Δ lethality. I found that mutations in the genes encoding RNA Polymerase III subunit Rpc17, sister chromatid cohesion protein Chl1, or the protein chaperone Hsp104 could rescue viability. Of these genes, the role of Hsp104 in yeast is best understood. Hsp104 plays a role in prion propagation, including the maintenance of [PSI+], which contributes to the synthesis of nonstop proteins. I demonstrate that rtf1Δ and rkr1Δ are synthetically lethal only in the presence of [PSI+]. The deletion, inactivation, and overexpression of HSP104 or the overexpression of prion-encoding genes URE2 and LSM4 clear [PSI+] and rescue rtf1Δ rkr1Δ lethality. In addition, the presence of [PSI+] decreases the fitness of rkr1Δ strains. I investigated whether the loss of RTF1 exacerbates an overload in nonstop proteins in rkr1Δ [PSI+] strains but, using reporter plasmids, found that rtf1Δ decreases nonstop protein levels, indicating that excess nonstop proteins may not be the cause of synthetic lethality. Although the mechanism of suppression is not yet clear, mutations in CHL1 suppress this rtf1Δ-specific defect by increasing nonstop protein levels. In addition, I have performed experiments to identify substrates and interacting partners for Rkr1 and these results have further indicated a role of Rkr1 in protein quality control. My data suggest that the loss of Rtf1-dependent histone modifications increases a burden on quality control in rkr1Δ [PSI+] cells. Importantly, my research indicates an essential connection between these conserved processes of transcription and quality control.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairArndt, Karenarndt@pitt.eduARNDT
Committee MemberBrodsky, Jeffreyjbrodsky@pitt.eduJBRODSKY
Committee MemberMartens, Joemartens@pitt.eduMARTENS
Committee MemberVanDemark, Andrewandyv@pitt.eduANDYV
Committee MemberOpresko, Pattyplo4@pitt.eduPLO4
Date: 27 September 2012
Date Type: Publication
Defense Date: 20 June 2012
Approval Date: 27 September 2012
Submission Date: 5 July 2012
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 219
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: RKR1, RTF1, Prions, Nonstop Protein, PAF1, Quality control
Date Deposited: 27 Sep 2012 23:45
Last Modified: 27 Sep 2017 05:15

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