Identification of Rkr1, a Nuclear RING Domain Protein with Functional Connections to Chromatin Modification in Saccharomyces cerevisiae

Abstract

Proper transcription by RNA polymerase II is dependent on the modification state of the chromatin template. The Paf1 complex is associated with RNA polymerase II during transcription elongation and is required for several histone modifications that mark active genes. To uncover additional factors that regulate chromatin or transcription, we performed a genetic screen for mutations that cause lethality in the absence of the Paf1 complex component Rtf1. Our results have led to the discovery of a previously unstudied gene, RKR1. Strains lacking RKR1 exhibit phenotypes associated with defects in transcription and chromatin function. These phenotypes include inositol auxotrophy, impaired telomeric silencing, and synthetic lethality with mutations in SPT10, a gene that encodes a putative histone acetyltransferase. In addition, deletion of RKR1 causes severe genetic interactions with mutations that prevent histone H2B lysine 123 ubiquitylation or histone H3 lysine 4 methylation. RKR1 encodes a conserved nuclear protein with a functionally important RING domain at its carboxy terminus. In vitro experiments indicate that Rkr1 possesses ubiquitin-protein ligase activity. Taken together, our results identify a new participant in a protein ubiquitylation pathway within the nucleus that acts to modulate chromatin function and transcription.Progression of the RNA polymerase II (Pol II) transcription cycle involves the coordinated functions of a large number of regulatory proteins. During transcription elongation, proteins that associate with Pol II assist it in overcoming obstacles to transcription, including DNA damage and condensed chromatin structure. Transcription elongation factors use a variety of mechanisms to facilitate movement of Pol II through a nucleosomal template and coordinate transcription with RNA processing. The Paf1 complex is a Pol II-associated factor that alters the state of the chromatin template during transcription elongation. Biochemical purification of the Saccharomyces cerevisiae Paf1 complex showed that it minimally contains five proteins: Paf1, Ctr9, Rtf1, Cdc73, and Leo1 (37,44,76). Chromatin immunoprecipitation experiments demonstrated that this complex is associated with the open reading frames (ORFs) of actively transcribed genes (37,59,72). Strains lacking members of the Paf1 complex are sensitive to the base analogs 6-azauracil and mycophenolic acid and exhibit altered RNA levels for a large number of genes (10,44,57,76). These results, combined with genetic and physical interactions with the elongation factors Spt4-Spt5 and Spt16-Pob3 (yFACT complex) (76), suggest that the Paf1 complex is important for transcription elongation. Members of the Paf1 complex are also required for proper 3Ј end formation of both polyadenylated and nonpolyadenylated transcripts (57, 68).It is now well established that the arrangements of posttranslational modifications on nucleosomal histones, along with interactions between histones and nonhistone proteins, coordinately affect chromatin structure. Histones can...

Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity

“…In Vitro Ubiquitination Assay-In vitro ubiquitination assays were performed as described (12). Briefly, purified S. pombe E1 (Uba1), E2 (Ubc4), wild-type Dsc1 RING domain (aa 608 -695), and Dsc1 mutants (C634A, H668A, I636D, and L675D) were used in this assay.…”

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

“…Serial dilution analysis was conducted on 5-FOA medium to select for cells that had lost the URA3-marked wild-type HTA1-HTB1 plasmid and retained the HIS3-marked HTA1-htb1K123R plasmid. As previously shown, the rkr1D mutation causes a strong synthetic growth defect in combination with the H2B K123R substitution by this assay ( Figure 2C) (Braun et al 2007). However, if these strains were first cured of prions by passaging on medium containing 5 mM GuHCl prior to plating on 5-FOA medium, rkr1D htb1K123R strains were viable ( Figure 2C).…”

“…Depletion of human Paf1C (hPaf1C) subunits impairs mRNA cleavage, polyadenylation, and (Braun et al 2007). Deletion of RKR1 also causes severe growth defects in strains lacking PAF1 or CTR9 (Braun et al 2007). Given that rkr1D causes severe synthetic growth defects in strains with an htb1-K123R mutation, Rkr1 most likely functions in a pathway parallel to the histone modification functions of Paf1C to promote an important cellular process (Braun et al 2007).…”

“…Rkr1 has been reported to interact with ribosomes and be localized in the cytoplasm, where it is then necessary for nonstop protein degradation (Fleischer et al 2006;Bengtson and Joazeiro 2010). Because our previous studies indicated nuclear localization of Rkr1 (Braun et al 2007), we decided to re-examine the localization of Rkr1 using different strains, an N-terminally tagged HA-Rkr1 construct, and better visualization using confocal microscopy. Here, we found that Rkr1 is predominantly, although not exclusively, cytoplasmic, thus supporting its role in nonstop protein degradation in our strains ( Figure S3).…”

“…Interestingly, the HSP104 high-copy plasmid allowed for growth of the rtf1D rkr1D double mutants ( Figure 2B). Taken together, our results indicate that deletion, inactivation, or overexpression of HSP104 suppresses rtf1D rkr1D synthetic lethality by clearing [PSI + ].A defect in H2B K123 ubiquitylation can phenocopy an rtf1D mutation with respect to rkr1D synthetic growth defects (Braun et al 2007). We therefore investigated if the inactivation of Hsp104 could also rescue the genetic interaction between rkr1D and htb1-K123R, a derivative of H2B that lacks the ubiquitylation site for Rad6-Bre1.…”