Functional interactions between the transcription and mRNA 3' end processing machineries mediated by Ssu72 and Sub1

Abstract

Transcription and processing of pre-mRNA are coupled events. By using a combination of biochemical, molecular, and genetic methods, we have found that the phylogenetically conserved transcription factor Ssu72 is a component of the cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae. Our results demonstrate that Ssu72 is required for 3' end cleavage of pre-mRNA but is dispensable for poly(A) addition and RNAP II termination. The in vitro cleavage defect caused by depletion of Ssu72 from cells can be rescued by addition of recombinant Ssu72. Ssu72 interacts physically and genetically with the Pta1 subunit of CPF. Overexpression of PTA1 causes synthetic lethality in an ssu72-3 mutant. Moreover, Sub1, which has been implicated in transcription initiation and termination, also interacts with Pta1, and overexpression of SUB1 suppresses the growth and processing defect of a pta1 mutation. Physical interactions of Ssu72 and Sub1 with Pta1 are mutually exclusive. Based on the interactions of Ssu72 and Sub1 with both the Pta1 of CPF and the TFIIB component of the initiation complex, we present a model describing how these novel connections between the transcription and 3' end processing machineries might facilitate transitions in the RNAP II transcription cycle.

Figure 1

Ssu72 is associated with the cleavage/polyadenylation factor (CPF). (A) Affinity purification of proteins associated with TAP-tagged Pta1. Protein was isolated from a yeast strain expressing TAP-tagged Pta1 by using IgG-agarose beads and eluted by TEV protease cleavage (lane 2). The complex was further purified by using calmodulin beads and eluted with EGTA (lane 3). Proteins were separated on a 10% polyacrylamide–SDS gel and stained with silver. The sizes of protein markers (lane 1) in kilodaltons are indicated on the left. (B) CPF subunits coimmunoprecipitate with Ssu72 from whole-cell extract of SSU72-td cells expressing Ubiquitin-Arg-DHFR^ts-HA-Ssu72. Proteins were immunoprecipitated with anti-HA (lanes 1,2) or anti-Pip1 (lanes 3,4) antibodies bound to protein A agarose beads. Immunoprecipitates (IP) and 10% of the supernatants (SN) were resolved by a 10% polyacrylamide–SDS gel. Proteins were detected with antibodies as indicated on the right.

Figure 2

Ssu72 interacts with Pta1 and is required for accurate cleavage of RNA14 mRNA in vivo. (A) Ssu72 interacts directly with Pta1. Recombinant GST-Ssu72 or GST alone was bound to glutathione-Sepharose beads and then incubated with proteins radioactively labeled by in vitro translation. The proteins bound to GST or GST-Ssu72 and 10% of the input were separated on a 10% polyacrylamide–SDS gel and detected by autoradiography. (B) Overexpression of PTA1 is lethal in combination with the ssu72-3 mutation. Isogenic SSU72 wild-type, ssu72-3, and ssu72-7 strains were transformed with the URA3 plasmids pYES (vector) or pYES-PTA1 (PTA1), from which PTA1 is expressed under the control of a galactose-inducible promoter. Purified transformants were streaked on synthetic medium lacking uracil (−URA) and containing either 2% glucose (−URA/Glc) or 2% galactose (−URA/Gal) as the sole carbon source and incubated for 2 d at 30°C. (C) Schematic representation of RNA14 gene and its transcripts. Arrows indicate the poly(A) sites. The position of the DNA fragment used as the probe in Northern blot analysis is indicated by a black bar. (D) Northern blot analysis of RNA14 mRNA showing poly(A)⁺ mRNAs from 100 μg of total RNA isolated from the ssu72-3 mutant (lanes 3,4) or isogenic wild-type SSU72 (lanes 1,2) cells grown at 25°C (lanes 1,3) or shifted for 90 min to 37°C (lanes 2,4). The approximate size of each transcript is indicated in kilobases on the left.

Figure 3

Ssu72 is required for in vitro 3′ end processing. (A) Coupled cleavage/polyadenylation assays. Extracts (70 μg) from ssu72 mutants (lanes 5–7,11–16) or the isogenic wild-type strains (lanes 2–4,8–10) were incubated with ATP and ³²P-labeled full-length GAL7-1 RNA (lane 1, precursor) for 20 min at 30°C. Products were resolved on a denaturing 5% polyacrylamide gel and visualized with a PhosphorImager. In some cases, extracts were heated at 37°C for the indicated times before adding to the reaction mixture. Positions of the precursor RNA and products are shown by the bars on the left. The downstream cleavage product is rapidly degraded and not visible. (B) Poly(A) addition assays. The assays were performed as in A except that precleaved RNA substrate GAL7-9 (lane 1) was used as precursor. (C) Complementation of processing activity in the heat-treated ssu72-3 mutant extract. Reactions were carried out as in A with extracts as indicated at the top of each lane. (D) Western blot analysis of whole-cell extracts prepared from ssu72 mutants and isogenic wild-type strains. Equal amounts of protein (70 μg) from wild type and ssu72 mutants as indicated on the top of each lane were separated on a 10% polyacrylamide–SDS gel and proteins detected with antibodies as shown on the left. In some cases, extracts were heated for 30 min at 37°C.

Figure 3

Ssu72 is required for in vitro 3′ end processing. (A) Coupled cleavage/polyadenylation assays. Extracts (70 μg) from ssu72 mutants (lanes 5–7,11–16) or the isogenic wild-type strains (lanes 2–4,8–10) were incubated with ATP and ³²P-labeled full-length GAL7-1 RNA (lane 1, precursor) for 20 min at 30°C. Products were resolved on a denaturing 5% polyacrylamide gel and visualized with a PhosphorImager. In some cases, extracts were heated at 37°C for the indicated times before adding to the reaction mixture. Positions of the precursor RNA and products are shown by the bars on the left. The downstream cleavage product is rapidly degraded and not visible. (B) Poly(A) addition assays. The assays were performed as in A except that precleaved RNA substrate GAL7-9 (lane 1) was used as precursor. (C) Complementation of processing activity in the heat-treated ssu72-3 mutant extract. Reactions were carried out as in A with extracts as indicated at the top of each lane. (D) Western blot analysis of whole-cell extracts prepared from ssu72 mutants and isogenic wild-type strains. Equal amounts of protein (70 μg) from wild type and ssu72 mutants as indicated on the top of each lane were separated on a 10% polyacrylamide–SDS gel and proteins detected with antibodies as shown on the left. In some cases, extracts were heated for 30 min at 37°C.

Figure 4

In vivo depletion of Ssu72 affects the cleavage step of pre-mRNA 3′ end processing. (A) The level of Pta1 decreases on Ssu72 depletion in vivo. Western blot analysis was performed with extracts from wild-type (WT, lane 1) or SSU72-td (lanes 2–5) cells grown at 25°C (lanes 1,2) or shifted to 37°C for the indicated times (lanes 3–5). Antibodies used are indicated on the right. (B) Depletion of Ssu72 in vivo affects in vitro 3′ end processing. The coupled cleavage/polyadenylation assay (lanes 2–5) was carried out as described in Figure 3A with the extracts (70 μg) from the SSU72-td strain described in A. Poly(A) addition assays (lanes 7–10) were performed as described in Figure 3B. Unreacted precursor (Pre) is shown in lanes 1 and 6. (C) Recombinant Ssu72-His6 can rescue the Ssu72-depleted extract. Extract (35 μg) from each Ssu72-depletion (lanes 3–5) was supplemented with 200 ng of Ssu72-His6 (lanes 6–8) or rna15-1 extract (lanes 10–12). (Lane 1) Unreacted precursor. (Lane 9) Activity of the rna15-1 extract.

Figure 5

The ssu72 mutants do not affect transcription termination. (A) Transcription run-on (TRO) analysis. Locations of the probes (C1–C6) and the poly(A) site on the CYC1 reporter gene are indicated. Yeast strains carrying the pCYC1 plasmid were grown at 25°C, or shifted to 37°C for 45 min before the TRO analysis. (B) Quantification of TRO data by PhosphorImager analysis. The signal from each probe was normalized according to the U content and corrected for the M13 background signal. The data were then normalized to the C1 probe, which was fixed at 100%. For SSU72 and ssu72-2, the results of two independent experiments were averaged. SSU72-td represents the average of three different experiments.

Figure 6

Sub1 physically and genetically interacts with Pta1. (A) Sub1 directly interacts with Pta1 and TFIIB but not Ssu72. For lanes 1–5, recombinant GST-Pta1, GST-Ssu72, GST-TFIIB, or GST alone (as indicated at the top of each lane) was bound to glutathione-Sepharose beads and incubated with in vitro translated [³⁵S]-Sub1. Ten percent of the Sub1 input (lane 1) and all of the bound proteins were separated on a 10% polyacrylamide–SDS gel. For lanes 6 and 7, [³⁵S]-Sub1 was incubated with anti-Pta1 bound to protein A agarose beads in the presence or absence of [³⁵S]-Pta1-His6. (B) Overexpression of SUB1 suppresses the pta1 thermo-sensitive phenotype in an allele-specific manner. The PTA1, pta1-2, and pta1-3 strains were transformed with a 2μ HIS3 SUB1 plasmid or vector (pRS423) alone. Transformants were purified on −His medium and incubated on rich medium at 30°C, 37°C, or 38.5°C for 2 d. Strains on the left side of each plate are derivatives of XH6 (PTA1) and XH15 (pta1-3); strains on the right side are derivatives of FY23 (PTA1) and FY1284 (pta1-2). (C) Deletion of SUB1 causes synthetic lethality in the pta1-2 mutant. Plasmid YCp-PTA1 was introduced into the pta1-2 mutant, and a resulting transformant was disrupted at the chromosomal SUB1 locus. The pta1-2 host strain (middle sector), pta1-2 mutant transformed with YCp-PTA1 (bottom sector), and its sub1Δ derivative (upper sector) were then streaked onto rich medium (YPD), synthetic medium lacking uracil (−URA), and synthetic medium that counter-selects the URA3 plasmid (5-FOA), and incubated at 25°C for 3–5 d. (D) Overexpression of SUB1 restores the processing activity of the pta1-2 mutant. Coupled cleavage/polyadenylation reactions were performed with extracts from wild type (WT, lane 2), pta1-2 (lane 3), pta1-2/vector (lane 4), and pta1-2/SUB1 (lane 5). Precursor RNA is shown in lane 1. Positions of the precursor RNA and polyadenylated products are indicated by the bars on the left.

Figure 7

Sub1 cannot bind to Pta1 in the presence of Ssu72. Recombinant GST-Ssu72 (lanes 3,6,7) or GST alone (lanes 2,5) was bound to glutathione-Sepharose beads and then incubated with in vitro translated Pta1 (lanes 2–3,7) and/or Sub1 (lanes 5–7). For lane 8, the same amount of in vitro translated Pta1 and/or Sub1 as used in lane 7 were incubated with anti-Pta1 antibody bound to protein A agarose beads. Proteins bound to the beads and 10% of the input of in vitro translated Pta1 (lane 1) and Sub1 (lane 4) were separated on a 10% polyacrylamide–SDS gel.

Figure 8

A model illustrating how Ssu72 and Sub1 might function at different points in the transcription cycle through interactions with TFIIB, RNAP II, cleavage/polyadenylation factor (CPF), and CF I. Based on the available data, the following scenario is possible, although several aspects need to be tested experimentally. (A) In the initiation stage, Ssu72 helps to correctly position RNAP II at the promoter through direct interactions with TFIIB and RNAP II (Sun and Hampsey 1996; Pappas and Hampsey 2000; Dichtl et al. 2002a). Ssu72 also recruits CPF to the promoter through its Pta1 partner and/or the weaker interactions with other CPF subunits. (B) Sub1 facilitates promoter clearance by its interaction with TFIIB, possibly by disrupting the Ssu72-TFIIB interaction as well as that of TFIIB and TFIID (Knaus et al. 1996). Sub1 may be brought to the promoter by CF I through an interaction with Rna15, or alternatively, CF I could displace Sub1 from TFIIB, so that reinitiation can occur. (C) Ssu72 and Sub1 act as positive elongation factors, as suggested previously for Ssu72 (Dichtl et al. 2002a) and Sub1 (Calvo and Manley 2001). (D) Recognition of processing signals by CPF and CF I triggers transcription termination, in part by releasing Sub1 from CF I and perhaps transferring it to CPF, where it stimulates cleavage by releasing Ssu72 from Pta1. The inset depicts the mutually exclusive interaction of Ssu72 and Sub1 with Pta1 during the transcription cycle. The C-terminal repeat domain of Rpb1 is depicted by the green extension from RNAP II that is extensively phosphorylated (P) in B–D. The blue line denotes pre-mRNA, capped at the 5′ end (C,D) and the 3′ processing site shown by p(A) in D.