The Paf1 Complex Subunit Rtf1 Buffers Cells Against the Toxic Effects of [PSI+] and Defects in Rkr1-Dependent Protein Quality Control in Saccharomyces cerevisiae

Abstract

The Rtf1 subunit of the Paf1 complex is required for specific 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 readthrough into the poly(A) tail. We performed a transposon-based mutagenesis screen to identify suppressors of rtf1D rkr1D lethality and found that a mutation in the gene encoding the protein chaperone Hsp104 rescued viability. Hsp104 plays a role in prion propagation, including the maintenance of [PSI + ], which contributes to the synthesis of nonstop proteins. We demonstrate that rtf1D and rkr1D 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 rtf1D rkr1D lethality. In addition, the presence of [PSI + ] decreases the fitness of rkr1D strains. We investigated whether the loss of RTF1 exacerbates an overload in nonstop proteins in rkr1D [PSI + ] strains but, using reporter plasmids, found that rtf1D decreases nonstop protein levels, indicating that excess nonstop proteins may not be the cause of synthetic lethality. Instead, our data suggest that the loss of Rtf1-dependent histone modifications increases the burden on quality control pathways in cells lacking Rkr1 and containing [PSI + ]. D URING transcription elongation, various proteins modify chromatin in coordination with RNA polymerase II (Pol II) to ensure accurate and efficient transcription of nucleosomal templates (Li et al. 2007). Changes in chromatin include nucleosome remodeling, the exchange of histone variants for canonical histones, and histone modifications such as the methylation, ubiquitylation, and acetylation of lysine (K) residues. The conserved Paf1 complex (Paf1C), which consists of Paf1, Ctr9, Leo1, Cdc73, and Rtf1, associates with Pol II on all actively transcribed genes (Mayer et al. 2010) and couples the modification of histones to transcription elongation (reviewed in Crisucci and Arndt 2011; Jaehning 2010). Paf1C is required for multiple histone modifications associated with active genes, including the monoubiquitylation of H2B K123, a modification for which the Rtf1 subunit of Paf1C plays a prominent role (Ng et al. 2003;Wood et al. 2003;Warner et al. 2007;Tomson et al. 2011). Rad6 is the ubiquitin-conjugating enzyme (E2) for H2B K123 ubiquitylation, while Bre1 is the ubiquitin-protein ligase (E3) (Hwang et al. 2003). This modification is a prerequisite for downstream histone H3 methylation (Dover et al. 2002;Sun and Allis 2002;Ng et al. 2003;Wood et al. 2003). In yeast, loss of H2B K123 ubiquitylation broadly impacts gene expression and chromatin structure (Mutiu et al. 2007;Batta et al. 2011). In humans, errors in Paf1C-dependent histone modifications can lead to aberrant gene expression and tumorigenesis (reviewed in Cris...

Interaction Networks of Prion, Prionogenic and Prion-Like Proteins in Budding Yeast, and Their Role in Gene Regulation

“…LSM4 directly interacts with PUB1, the third KP/EPD hub, and with two proteins (PUF4 and SCD6) predicted to be prionogenic by PrionScan [15] . Upon overexpression, LSM4 has been shown to form amyloids that play a key role in elimination of the [PSI+] prion from cells [42] – [44] . Also, ‘P-bodies’, which are cytoplasmic RNA granules that contain translationally repressed ribonucleoproteins, can be formed via the N/Q-rich domain of LSM4 [45] .…”

The Nucleosome Acidic Patch Regulates the H2B K123 Monoubiquitylation Cascade and Transcription Elongation in Saccharomyces cerevisiae

“…Rkr1 is a protein quality control factor that is involved in the degradation of aberrant proteins, including those that extend past stop codons [ 25 , 26 ]. The elevated synthesis of aberrant proteins, potentially as a consequence of improper transcription in the H2A mutants, could have lethal consequences for the cell [ 72 ]. The negative genetic interaction between rkr1∆ and the histone mutants suggests that the consequences of disrupting the acidic patch extend beyond chromatin and transcription.…”

Amyloids and Yeast Prion Biology

“…[PSI+] decreases the fitness of rkr1Δ cells defective in protein quality control (158). Certain mutations in the Hsp40 Sis1p do not impair its essential function but do result in [PSI+] being lethal for the cell (117) MOD5 encodes a tRNA isopentenyltransferase, and mod5Δ mutations slow cell growth but make them resistant to antifungal drugs of the fluconazole group that block ergosterol biosynthesis (162).…”

“…[PSI+] decreases the fitness of rkr1Δ cells defective in protein quality control. 158 Certain mutations in the Hsp40 Sis1p do not impair its essential function but do result in [PSI+] being lethal for the cell. 117 The combined presence of [PSI+] and [PIN+] is lethal in sla2Δ cells.…”