A Requirement for the Saccharomyces cerevisiae Paf1 complex in snoRNA 3' end formation

Abstract

RNA synthesis and processing are coordinated by proteins that associate with RNA polymerase II (pol II) during transcription elongation. The yeast Paf1 complex interacts with RNA pol II and mediates histone modifications during elongation. To elucidate the functions of this complex, we isolated missense mutations in the gene encoding the Rtf1 subunit and used them to identify functionally interacting proteins. We identified NAB3 as a dosage suppressor of rtf1. Nab3, together with Nrd1, directs 3' end formation of nonpolyadenylated RNA pol II transcripts, such as snoRNAs. Deletion of Paf1, but not the Set1, Set2, or Dot1 histone methyltransferases, causes accumulation of snoRNA transcripts that are extended at their 3' ends. The Paf1 complex associates with and facilitates Nrd1 recruitment to the SNR47 gene, suggesting a direct involvement in 3' end formation. Our results reveal a posttranscriptional function for the Paf1 complex, which appears unrelated to its role in histone methylation.

Figure 1. Characterization of rtf1 Point Mutants

(A) Equivalent numbers of cells from wild-type (wt) (RTF1) (KY767), rtf1-105 (KKY64), rtf1-100 (KKY56), rtf1-107 (KKY58), and rtf1Δ (KY656) strains were spotted in 10-fold serial dilutions onto the indicated media. Sensitivity to 6AU was examined on SC-Ura medium supplemented with 50 μg/ml 6AU (5 days of incubation). The Spt⁻ phenotype was analyzed on medium lacking histidine (3 days of incubation). (B) Immunoblot analysis was performed on wt (KY406), rtf1-105 (V274D; KKY37), rtf1-100 (Q172R; KKY40), rtf1-107 (M289K; KKY45), and rtf1Δ (KY410) cells. Cultures were grown to early log phase, divided, and further incubated at 30°C or 37°C for 2 hr. Extract from 1 × 10⁷ cells per sample was analyzed by immunoblotting with anti-Rtf1 and, as a loading control, anti-L3 antisera. (C) Histone H3 K4 trimethylation was analyzed by immunoblotting of whole-cell extracts prepared from strains grown as in (B).

Figure 2. Paf1 Complex Members Genetically Interact with NAB3 and NRD1

(A) Vector (pRS425), the original 2μ library plasmid containing NAB3, and a NAB3 sub-clone (2μ NAB3) were transformed into wt (KY767), rtf1Δ (KY656), and rtf1-107 (KKY58) strains. Suppression of the Spt⁻ phenotype was analyzed by spotting equivalent numbers of cells in 10-fold serial dilutions onto selective medium lacking histidine (5 days incubation). (B) A plasmid shuffle experiment was performed by transforming pRS314, pRS314-NRD1, or pRS314-nrd1-5 into nrd1Δ RTF1(KKY129), nrd1Δ rtf1Δ (KKY128), or nrd1Δ cdc73Δ (KKY143) cells that contained pRS316-NRD1. Transformants were passaged on 5-FOA to select against the pRS316-NRD1 plasmid. Equivalent numbers of cells were spotted in 10-fold serial dilutions onto YPD (4 days incubation).

Figure 3. SNR13 Readthrough Transcripts Accumulate in Paf1 Complex Mutant Strains

(A) Top, SNR13-TRS31 genomic locus and transcription products. The transcriptional readthrough product is indicated as SNR13-TRS31. (A–D) Northern analysis using a TRS31 probe and RNA isolated from the following yeast strains: (A) wt (KY661), rtf1Δ (KY656), paf1Δ (KY685), ctr9Δ (GHY1094), cdc73Δ (KY689), leo1Δ (GHY250), and ctk1Δ (KY586); (B) wt (FY118), spt4Δ (GHY166), spt5-194 (KY718), spt5-242 (FY1635), fcp1-110 (PCY448), and ctk1Δ (KY586); (C) SPT16 (FY118) and spt16-197 (FY348); and (D) wt (FY118), set1Δ (KY910), set2Δ (KY912), and dot1Δ (KY903). For each strain in (C), one-half of a log phase culture was shifted to 39°C for 80 min to inactivate the spt16-197 gene product (Kaplan et al., 2003). The remaining culture was kept at 30°C for 80 min.

Figure 4. Deletion of Paf1 Complex Members Impairs snR47 3′ End Formation

(A) Top, the SNR47 genomic locus and transcripts detected in wt cells or mutants defective in snR47 3′ end formation (SNR47-YDR042c). Transcription of YDR042c is not detected in wt cells. Bottom, Northern analysis on RNA from wt (FY118), rtf1Δ (KY656), paf1Δ (KY685), ctr9Δ (GHY1094), cdc73Δ (KY689), leo1Δ (GHY250), and nrd1-5 cells. RNA samples were loaded in duplicate on the same gel, one-half of which was subjected to Northern analysis with a YDR042c probe and the other half with an SNR47 probe. The positions of ethidium-bromide-stained RNA molecular weight markers are shown. Filters were reprobed for SCR1 transcript levels as a loading control. (B) Top, plasmids used in the reporter assay to monitor snR47 3′ end formation. Bottom, RTF1 (KY669) and rtf1Δ (KY560) cells transformed with the LEU2-marked plasmids AHC and ASHC were plated in 10-fold serial dilutions onto SC-His-Leu and SC-Leu media (2 days incubation at 30°C).

Figure 5. RNA Pol II and Paf1 Colocalize to the SNR47 Gene

(A) SNR47 locus with PCR products amplified in ChIP experiments. (B) Chromatin from wt (KY669) cells was immunoprecipitated with 8WG16 antibody. Two amounts of immunoprecipitated (IP) DNA (2 μl and 4 μl) and input (In) DNA (4 μl of 1:125 and 1:250 dilutions) were analyzed by PCR. The reactions included the SNR47 primer pair specified below each panel (arrow denotes PCR product) and a primer pair to an intergenic region of chromosome VIII ([Ng et al., 2003b]; asterisk denotes PCR product). (C) SNR47 ChIP signals were normalized to chromosome VIII signals. Normalized IP values were divided by normalized input values, and the ratios from three independent ChIP experiments were averaged. (D) ChIP was performed on chromatin prepared from HA₃-PAF1 (GHY972) cells with α-HA antibody. (E) Quantitation of three HA₃-Paf1 ChIP experiments was performed as in (C). Error bars in (C) and (E) indicate the SEM.

Figure 6. Paf1 Is Required for Normal Nrd1 Association with SNR47

(A) ChIP was performed on chromatin prepared from PAF1 (KKY120) and paf1Δ (KKY122) cells with α-Nrd1 antibody (IP) or preimmune sera (Pi). (B) Quantitation of three ChIP experiments as in (A). Relative ChIP signals, calculated as in Figure 5, are shown for Nrd1 in the presence (black bars) or absence (white bars) of Paf1. (C) Protein extracts, prepared from cells grown for ChIP as in (A), were analyzed by immunoblotting with α-Nrd1 and α-L3 antibodies. (D) ChIP analysis was performed on chromatin from PAF1 HA-RPB3 (GHY645) and paf1Δ HA-RPB3 (KY832) cells. Black and white bars represent average enrichment of HA-Rpb3 in PAF1 and paf1Δ cells, respectively. Untagged control strains (FY118 and KY685) yielded ChIP signals that were near background levels (not shown). Error bars in (B) and (D) indicate the SEM.