Leo1 subunit of the yeast paf1 complex binds RNA and contributes to complex recruitment

Abstract

The Paf1 complex (Paf1C) affects RNA polymerase II transcription by coordinating co-transcriptional chromatin modifications and helping recruit mRNA 3' end processing factors. Paf1C cross-links to transcribed genes, but not downstream of the cleavage and polyadenylation site, suggesting that it may interact with the nascent mRNA. Paf1C purified from Saccharomyces cerevisiae binds RNA in vitro, as do the purified Leo1 and Rtf1 subunits of the complex. In vivo cross-linking and immunoprecipitation of RNA associated with Paf1C (RNA-IP) show that Leo1, but not Rtf1, is necessary for the complex to bind RNA. Cells lacking Leo1 have reduced Paf1C recruitment as well as decreased levels of histone H3 and trimethylated H3 Lys(4) within transcribed chromatin. Together, these results suggest that association of Paf1C with RNA stabilizes its localization at actively transcribed regions where it influences chromatin structure.

FIGURE 1.

Paf1 complex binds RNA in vitro. A, RNA EMSA was performed using purified Paf1C and radiolabeled pBlue RNA. Concentrations of Paf1C are indicated above the gel, and the asterisk shows the position of the free probe. B, RNA EMSA reactions containing radiolabeled pBlue RNA and 28 nm Paf1C were incubated with increasing concentrations (0.2–2 μm) of unlabeled competitor RNA. The pBlue competitor is the same sequence as the radiolabeled probe whereas the GAL7 competitor is a completely different sequence. C, Paf1C RNA binding was challenged with unlabeled competitor dsDNA that contains the same sequence as the radiolabeled RNA probe. DNA was titrated from 0.05 to 2 μm. D and E, Paf1C binding was challenged with unlabeled competitor ssDNA. DNA was titrated from 0.05 to 5 μm in D and 0.05 to 2 μm in E. The T3 promoter primer oligonucleotide (19 nucleotides) was used in D and an oligonucleotide corresponding to the coding region of Rpb1 (88 nucleotides) was used in E. For all gels, the asterisk denotes migration of the unbound RNA probe. Lanes marked Paf1C indicate reactions containing 28 nm Paf1C with no unlabeled competitor, and lanes marked with (−) indicate reactions lacking Paf1C.

FIGURE 2.

Leo1 and Rtf1 bind RNA in vitro. A, RNA binding reactions were performed as for Fig. 1A using radiolabeled RNA and the indicated concentrations of TAP-purified Paf1C. Reactions were cross-linked using UV irradiation, and unbound RNA was removed by RNase digestion. Radioactively labeled proteins were resolved by SDS-PAGE and visualized by phosphorimaging. B–E, RNA EMSA was performed using radiolabeled RNA and recombinant Cdc73 (B), Leo1 (C), Paf1 (D), or Rtf1 (E). Proteins were incubated with RNA in increasing concentrations as indicated above each lane. Asterisks denote migration of the unbound RNA. The (−) indicates reactions with the RNA probe alone.

FIGURE 3.

Paf1 complex lacking Rtf1 retains RNA binding activity. A, RNA EMSA was performed as in Fig. 1A using TAP-purified Paf1C isolated from either a wild-type or rtf1Δ strain. A single-shifted band (white arrowhead) is observed in reactions containing Paf1C(rtf1Δ) compared with the doublet seen in reactions with wild-type Paf1C. B, RNA EMSA was performed using radiolabeled RNA and Paf1C(rtf1Δ), and recombinant Rtf1 was added in increasing concentrations from 0.1 to 2 μm as indicated above the gel. Addition of Rtf1 led to the formation of a Paf1C·RNA complex that included Rtf1, which migrates similarly to the upper band of the wild-type Paf1C·RNA doublet (black arrow). The black arrowhead indicates formation of Rtf1·RNA complexes. The asterisks denote migration of the unbound RNA.

FIGURE 4.

Paf1C cross-links to mRNA transcripts. RNA-IP was performed for the YEF3, PYK1, ADH1, and PMA1 mRNAs using strains expressing the indicated TAP-tagged proteins. Proteins and associated RNA were precipitated with IgG-agarose (TAP IP) or anti-Rpb3 (Rpb3 IP) and analyzed using RT-PCR (IP). All five subunits of Paf1C co-precipitate RNA from all tested genes (lanes 3–7). Paf1 association with RNA is decreased in a leo1Δ strain (lane 8) but not in an rtf1Δ strain (lane 9). IP from an untagged strain (No tag; YF336) is shown as a negative control, and IP from an Rpb3-TAP (YF924) strain is shown as a positive control (lanes 1 and 2). Rpb3 association with RNA is unchanged in both a leo1Δ strain and an rtf1Δ strain (compare lane 10 with lanes 11 and 12). RT-PCR from total RNA is shown as the Input.

FIGURE 5.

Paf1 occupancy is reduced at actively transcribed genes in a leo1Δ strain. A, schematic representation of the YEF3 and PYK1 genes is shown, with numbered bars above the genes representing the PCR products analyzed by ChIP (see supplemental Table S2). Numbers below genes represent nucleotide positions of the open reading frame beginning at +1. B, ChIP was performed on the YEF3 and PYK1 genes using wild-type and leo1Δ strains expressing TAP-tagged Paf1. Rpb3 levels from the same chromatin preparation were also assayed by ChIP. The left panel shows the PCR products from both genes, and the right panels show quantification of the results. The precipitated protein is indicated above the gels. The input control was used to normalize the PCR products from the immunoprecipitation. The asterisk indicates an internal background control from a nontranscribed region of chromosome VI. The occupancy value is the ratio of the specific primer products to the internal negative control after normalizing to the input. The values shown represent the average, and the bars indicate S.D. for three independent replicates.

FIGURE 6.

RNA transcript contributes to Paf1 recruitment. A, B, and D, ChIP was performed for TAP-tagged Paf1 (A) as well as Rpb3 (B) and TBP (D) in wild-type (YF862) and leo1Δ (YSB2176) strains using the same chromatin preparation with or without RNase treatment prior to immunoprecipitation. PCR products are numbered, and occupancy values are calculated as for Fig. 5. C, levels of Paf1-TAP were normalized to the levels of Rpb3 at both YEF3 and PYK1.

FIGURE 7.

Loss of Leo1 alters H3 occupancy and H3K4 trimethylation. A, H3K4me3 levels are reduced in a leo1Δ strain. ChIP for H3K4me3 and H3 was performed in wild-type (YF336) and leo1Δ (YF347) strains. The upper panel shows absolute levels of H3K4me3 at YEF3 and PYK1, normalized to a nontranscribed region. The lower panel shows levels for H3K4me3 on both genes normalized to total H3. Note that the high level of H3K4me3 at the 3′ end for YEF3 is due to the proximity of this primer pair to the promoter of the downstream gene. B, H3K36me3 levels are maintained in a leo1Δ strain. ChIP for H3K36me3 and H3 was performed in wild-type and leo1Δ strains. The levels for H3K36me3 were normalized to a nontranscribed region (upper panel) and to total H3 levels (lower panel). C, H3 occupancy is decreased in a leo1Δ strain as determined by ChIP when normalized to a nontranscribed region. All values are shown with S.D. for three independent experiments. Quantification was done as for Fig. 6.