Effects of the Paf1 complex and histone modifications on snoRNA 3'-end formation reveal broad and locus-specific regulation

Abstract

Across diverse eukaryotes, the Paf1 complex (Paf1C) plays critical roles in RNA polymerase II transcription elongation and regulation of histone modifications. Beyond these roles, the human and Saccharomyces cerevisiae Paf1 complexes also interact with RNA 3'-end processing components to affect transcript 3'-end formation. Specifically, the Saccharomyces cerevisiae Paf1C functions with the RNA binding proteins Nrd1 and Nab3 to regulate the termination of at least two small nucleolar RNAs (snoRNAs). To determine how Paf1C-dependent functions regulate snoRNA formation, we used high-density tiling arrays to analyze transcripts in paf1Δ cells and uncover new snoRNA targets of Paf1. Detailed examination of Paf1-regulated snoRNA genes revealed locus-specific requirements for Paf1-dependent posttranslational histone modifications. We also discovered roles for the transcriptional regulators Bur1-Bur2, Rad6, and Set2 in snoRNA 3'-end formation. Surprisingly, at some snoRNAs, this function of Rad6 appears to be primarily independent of its role in histone H2B monoubiquitylation. Cumulatively, our work reveals a broad requirement for the Paf1C in snoRNA 3'-end formation in S. cerevisiae, implicates the participation of transcriptional proteins and histone modifications in this process, and suggests that the Paf1C contributes to the fine tuning of nuanced levels of regulation that exist at individual loci.

Fig 1

snoRNAs require Paf1 for proper 3′-end formation. (A) Depiction of regions downstream of the snoRNA genes that were used to design probes for Northern blot analysis (black bar) or amplification for RT-qPCR analysis (white bar). See Table 2 for details of all primers used. (B to D) Representative Northern blot analyses of extended snoRNA in wild-type cells (strains KY1699 [B], KY2278 [C], and KY2048[D]) and paf1Δ cells (strains KY1700 [B], KY2279 [C], and KY2279 [D]). Extended snoRNA transcripts were detected with probes to the intergenic region between the indicated snoRNA gene and the downstream ORF. SCR1 transcript levels serve as a loading control. (E) Quantification of extended SNR47, SNR48, and SNR79 transcript levels performed by Northern blot analysis with the relative signals of wild-type cells set at 1 as described in Materials and Methods. The SEMs of paf1Δ samples are indicated by the error bars. (F) Quantification of transcript levels in the regions between SNR47-YDR042C, SNR85-NUP188, and SNR32-CHS7 in wild-type (KY2278) and paf1Δ (KY2279) cells as measured by RT-qPCR, with the relative signal of wild-type cells set at 1 as described in Materials and Methods. The SEMs are indicated by the error bars.

Fig 2

Subunits of the Paf1 complex demonstrate a range of contributions to snoRNA 3′-end formation. Northern blot analyses were performed using RNA from wild-type (WT) (KY1699), paf1Δ (KY1700), ctr9Δ (KY1705), cdc73Δ (KY1706), rtf1Δ (KY1704), and leo1Δ (KY1805) cells. SCR1 transcript levels serve as a loading control. For quantification, the relative signal of wild-type cells was set at 1 (as described in Materials and Methods) and the SEMs are indicated by the error bars. (A) Representative Northern blot analysis and the quantification of extended SNR47 transcripts (SNR47-YDR042C). (B) Representative Northern blot analysis and the quantification of extended SNR48 transcripts (SNR48-ERG25).

Fig 3

snoRNAs require Rad6 but exhibit differential requirements for Rtf1-mediated H2B K123 monoubiquitylation. (A) Quantification of extended SNR47 (SNR47-YDR042C), SNR85 (SNR85-NUP188), and SNR32 (SNR32-CHS7) transcript levels in wild-type (KY2048), rtf1Δ (KY2277), and bre1Δ (KY2046) cells as determined by RT-qPCR, with the relative signal of wild-type cells set at 1. The SEMs are indicated by the error bars. Values that are significantly different (P value of <0.05) from the wild-type value are indicated by an asterisk. (B) Representative Northern blot analysis of extended SNR48 transcripts (SNR48-ERG25) in wild-type (KY1699), paf1Δ (KY1700), rtf1Δ (KY2041), bre1Δ (KY1713), rad6Δ (KY1712), hta2Δ htb2Δ (KY2043), and htb1-K123R hta2Δ htb2Δ (KY2044) cells. SCR1 transcript levels serve as a loading control. (C) Quantification of SNR48-ERG25 transcript levels performed as described above for panel B. For the four strains on the left side of the graph, the relative signal of wild-type (KY1699) cells was set at 1. The transcript levels in htb1-K123R hta2Δ htb2Δ cells (graph, right side) were made relative to the levels in hta2Δ htb2Δ (KY2043) cells. The SEMs are indicated by error bars. (D) Quantification of Northern blot analyses of extended SNR48 transcripts (SNR48-ERG25) in paf1Δ (KY1700), rad6Δ (KY1712), and paf1Δ rad6Δ (KY1378) cells. The relative signal of paf1Δ was set at 1, and the SEMs are indicated.

Fig 4

Paf1 is required for proper transcription termination at snoRNAs. (A and B) ChIP analysis of Rpb3 occupancy in wild-type (KY1699) and paf1Δ (KY1700) cells. A no-antibody control (No Ab) was performed using wild-type (KY1699) cells. Quantification depicts average occupancy in three independent experiments as described in Materials and Methods. The SEMs are indicated by error bars, and asterisks indicate a P value of <0.05 relative to the value for the wild type. Rpb3 occupancy was examined near SNR47 (A) and SNR48 (B) at indicated locations (see Table 2 for primers used). (C and D) Quantification of Northern blot analyses of the extended SNR47 (C) and SNR48 (D) transcripts in paf1Δ (KY1700), rrp6Δ (KY1267), and paf1Δ rrp6Δ (KY2377) cells. The relative signal of rrp6Δ was set at 1, and the SEMs are indicated.

Fig 5

Bur2 is required for proper snoRNA 3′-end formation. (A and B) Northern blot analyses were performed using RNA from wild-type (KY1699) and bur2Δ (KY1718) cells. SCR1 transcript levels serve as a loading control. For quantification, the relative signal of wild-type was set at 1. The SEMs of bur2Δ samples are indicated by the error bars. Representative Northern blot analysis and quantification of extended SNR47 (A) and SNR48 (B) transcripts are shown. (C and D) ChIP analysis of Rpb3 occupancy in wild-type (KY1699) and bur2Δ (KY1718) cells, which was examined near SNR47 (C) and SNR48 (D) at the indicated locations (see Table 2 for primers used). Quantification depicts average occupancy in three independent experiments. A no-antibody control (No Ab) was performed using wild-type (KY1699) cells. The SEMs are indicated by error bars, and asterisks indicate a P value of <0.05 relative to the value for the wild type. (E and F) Quantification of Northern blot analyses of extended SNR47 (E) and SNR48 (F) transcripts in paf1Δ (KY1700), bur2Δ (KY1718), and paf1Δ bur2Δ (KY1453) cells. The relative signal of paf1Δ was set at 1, and the SEMs are indicated.

Fig 6

Analysis of SNR47 and SNR48 read-through transcripts. (A and B) As described in Materials and Methods, strand-specific cDNA was generated using a primer downstream of either SNR47 or SNR48 (primer locations indicated by the arrows pointing left). For a control, a no-reverse transcriptase (no-RT) reaction was performed on each sample as indicated by the minus sign. PCRs were done with two different concentrations of each cDNA using a primer within the snoRNA sequence and a primer downstream of the snoRNA (sequences found in PCR product indicated by a black bar). ACT1 is used as a positive control for RT-PCR experiments. RT-PCR analysis was performed in triplicate from independent biological replicates. See Table 2 for the sequences of the primers used. (A) Representative RT-PCRs at SNR48 from wild-type (KY1699), paf1Δ (KY1700), rad6Δ (KY2045), bur2Δ (KY1718), and rtf1Δ (KY1703) cells. (B) Representative RT-PCRs at SNR47 from wild-type (KY1699), paf1Δ (KY1700), rad6Δ (KY2339), bur2Δ (KY1718), and rtf1Δ (KY1703) cells. (C) Northern blot analysis using RNA from wild-type (KY2276), paf1Δ (KY1702), and bur2Δ (KY2409) cells. Identical RNA was run on the same gel in parallel, and then after the gel was transferred to a membrane, each half of the membrane was hybridized with either a probe against the snoRNA sequence of SNR47 or a probe against the regions downstream of SNR47 (SNR47-YDR042C). Both probes recognized the SNR47 read-through transcript depicted.

Fig 7

Set2 is involved in snoRNA termination through a pathway distinct from H2B ubiquitylation. (A) Representative Northern blot analysis of extended SNR47 transcripts (SNR47-YDR042C) in wild-type (KY2090) and set2Δ (KY2280) cells. SCR1 transcript levels serve as a loading control. (B) Quantification of SNR47-YDR042C transcript levels in wild-type (KY2048), set2Δ (KY2282), and set2(1-261) (KY2281) cells measured by RT-qPCR, with the relative signal of the wild type set at 1. The SEMs are indicated by the error bars. Asterisks indicate a P value of <0.0025 relative to wild type. (C) The following his3Δ200 leu2Δ1 strains were transformed with an established LEU2-marked SNR47 reporter plasmid [pADH1-SNR47(70)-HIS3-CYC1] or control plasmid (pADH1-HIS3-CYC1) (see Materials and Methods for more information): wild-type (KY2042), paf1Δ (KY1664), rtf1Δ (KY2041), set2Δ (KY2280), and set2(1-261) (KY2338). Tenfold serial dilutions of cells containing the listed plasmids were spotted on synthetic complete medium lacking leucine (SC-leu) and synthetic complete medium lacking leucine and histidine (SC-leu-his) and incubated for 5 days at 30°C. (D) Representative Northern blot analysis examining HIS3 transcripts from the SNR47 reporter plasmid in his3Δ200 leu2Δ1 strains (as in panel C). Wild-type cells with the control plasmid (pADH1-HIS3-CYC1) show the size of the HIS3 transcript lacking 70 bp of the SNR47 sequence. LEU2 transcript levels serve as a control for plasmid levels, and SCR1 transcript levels control for total RNA. RNA from a his3Δ200 leu2Δ1 strain (KY2090) containing no plasmids (WT) (lane 2) serves as a control for probe specificity. (E) Quantification of SNR47 extended transcripts (SNR47-YDR042C) in wild-type (KY2048), set2Δ (KY2282), bre1Δ (KY2046), and set2Δ bre1Δ (KY2283) cells determined by RT-qPCR, with the relative signal of the wild type set at 1. The SEMs are indicated by the error bars.