Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

YHH1 is required for ACT1 poly(A) site recognition

To correlate the observed in vitro phenotypes with the lethality of yhh1 cells in vivo, we performed northern blot analysis on total RNA extracted from strains grown at 23°C and after shift to 37°C. Probes specific for ADH1, CYH2 and CYC1 revealed mRNA levels in yhh1 mutants grown at 37°C for up to 3 h, which were comparable to those in wild-type cells (Figure 2A, lanes 1–10). Some reduction in mRNA levels was observed in the mutants only after 4 h at 37°C. rna15–1 cells, in contrast, showed a strong reduction of these mRNAs after 1 h at 37°C (lanes 11 and 12). Levels of the very stable PGK1 mRNA (t_½ = 45 min) were similar in all strains tested. STE3 mRNA, which is very unstable (t_½ = 4.2 min), was strongly reduced in both wild-type and mutant YHH1 cells grown at 37°C; this RNA could not be detected in rna15–1 cells due to their mating type. Levels of 18S rRNA and U14 snoRNA served as loading controls and were comparable in all lanes. Interestingly, we observed that ACT1 mRNA was strongly reduced in yhh1-6 cells at early time points and also showed a smear of higher molecular weight species (lanes 7–10). The latter effect was more pronounced in yhh1-3 cells (lanes 3–6); rna15–1 cells showed a complete loss of ACT1 mRNA at 37°C (lane 12).

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Fig. 2. yhh1 mutant cells are defective in recognition of the ACT1 poly(A) site. (A) Northern analysis of total RNA extracted from wild-type and mutant YHH1 cells grown at 23°C, or following a shift to 37°C for 1.5, 3 and 4 h. For control, an rna15-1 strain grown at 23°C and 37°C was analysed in parallel. RNAs were separated on formaldehyde/1.2% agarose gels (panels i–v) or 8.3 M urea/8% polyacrylamide gels (panels vi–viii). Filters were developed with random-primed labelled probes or end-labelled oligonucleotides directed against the RNA species indicated on the right of each panel. (B) Analysis of ACT1 poly(A) site usage in wild-type and yhh1 and rna15-1 mutant cells as indicated. Total RNAs extracted from strains grown as described in (A) were treated with RNase H and oligonucleotide ACT1-RNaseH, and analysed by northern blotting. A random-primed probe complementary to the ACT1 3′-end region (nucleotides 930–1536) was used. The positions of RNAs polyadenylated at sites I to IV are indicated, as well as molecular weight markers. (C) Poly(A) tail analysis with total RNA extracted from indicated YHH1 strains after growth at 23°C (0 h) or after shift to 37°C for 1.5, 3 and 4 h. Also analysed was an rna15-1 strain grown at 23°C or after shift to 37°C for 1 h. Poly(A) tail length is indicated on the left. Strong signals of short RNA species in lanes 2, 4, 5, 6, 9, 10 and 12 were not reproducible and probably result from incomplete digestion of non-poly(A) sequences.

To resolve ACT1 mRNAs better, we treated total RNAs with RNase H targeted by an oligonucleotide complementary to the 3′ region of the message (ACT1-RNaseH). RNAs were analysed by polyacrylamide northern blot with a probe specific for the 3′-end of ACT1 mRNA. As shown in Figure 2B, at least four distinct ACT1 mRNA species (ACT1-I to -IV; lanes 1 and 2) can be distinguished, which originate from the use of alternative poly(A) sites (Mandart and Parker, 1995). Wild-type cells mainly use the most proximal poly(A) site (ACT1-I) and, to a lesser extent, upstream sites (ACT1-II to -IV). The same pattern of poly(A) site selection was observed in yhh1 mutant cells at 23°C; however, after shift to 37°C, the mutant cells increasingly used minor upstream sites (sites III and IV) at the expense of the major downstream site (site I; lanes 3–8).

Analysis of the global poly(A) tail length distribution revealed that yhh1 mutant strains did not show loss of poly(A) tails when grown at 37°C for up to 4 h (Figure 2C, lanes 3–10; data not shown for yhh1-12); in contrast, an rna15–1 strain showed a marked loss of poly(A) tails after shift to 37°C for 1 h (lanes 11 and 12) (Minvielle-Sebastia et al., 1994). Since yhh1 mutant strains ceased growth ∼200 min after shift to 37°C, this indicated that lethality of yhh1 mutants was not due to a general under-accumulation of mRNA. Instead, we propose that the formation of a subset of mRNAs with weak poly(A) sites requires Yhh1p for poly(A) site recognition (see Discussion). In agreement with the observed stability of mRNAs in yhh1 mutant cells, we did not see a reduction of Yhh1p or other tested 3′-end formation factors (Rna15p, Rna14p, Pcf11p, Ysh1p, Fip1p and Pap1p) by western analysis of extracts prepared from strains following growth at 37°C for up to 4 h (results not shown).

Yhh1p is an RNA-binding protein

Aberrant selection of ACT1 poly(A) sites suggested that Yhh1p might interact directly with the pre-mRNA. To test for RNA-binding activity, we performed glutathione S-transferase (GST) pull-down experiments with Yhh1p that was expressed in Escherichia coli with a GST tag fused to the N-terminus and a His₆ tag at the C-terminus (GST-Yhh1p-H₆; Figure 3A, lane 1). The identity of the purified 183 kDa protein was confirmed by western blot with an anti-Yhh1p antibody (Figure 3A, lane 2) (Stumpf and Domdey, 1996). Figure 3B shows that CYC1 RNA was bound efficiently (lane 4). CYC1-Pre RNA, which lacked sequences downstream of the poly(A) site and CYC1-512 RNA, which has a 38 nucleotide deletion encompassing both the efficiency element and positioning element, were bound efficiently as well (lanes 7 and 10). Wild-type GAL7-1 and GAL7-3 RNA, which has a deletion in the efficiency element, were bound more weakly compared with CYC1 substrates (lanes 13 and 16). The observed interactions were specific for the substrate RNA since reactions contained excess unlabelled E.coli tRNA. No binding could be observed when GST or GST-Ysh1p-H₆ was used (lanes 3, 6, 9, 12 and 15; results not shown).

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Fig. 3. Yhh1p is an RNA-binding protein. (A) Purified GST-Yhh1p-H₆ expressed in E.coli was subjected to SDS–PAGE and visualized by Coomassie Brilliant Blue staining (lane 1) or detected after western transfer with an anti-Yhh1p serum (lane 2). Positions of recombinant protein and marker bands are indicated. (B) GST pull-down experiments with 0.5 µg GST (lanes 3, 6, 9, 12 and 15) or GST-Yhh1p-H₆ (Yhh1p; lanes 4, 7, 10, 13 and 16) and in vitro transcribed, ³²P-labelled RNAs. Lanes 2, 5, 8, 11 and 14 show 10% of RNA included in binding reactions (Input). Length of marker bands (M; lane 1) is indicated on the left. (C) Sequence of sCYC1 RNA. Only CYC1-derived nucleotides are shown; vector encoded nucleotides are indicated by numbers in parentheses. Efficiency (EE) and positioning elements (PE), and upstream (UUE) and downstream (DUE) U-rich elements are underlined. The arrows mark poly(A)-site positions. Sequences complementary to 14-mer DNA oligonucleotides (1–8) are indicated. (D) RNase H protection analysis with Yhh1p and sCYC1 RNA. Internally ³²P-labelled sCYC1 RNA was incubated with binding buffer (No protein; lanes 2–11) or GST-Yhh1p-H₆. The GST moiety was removed from recombinant protein by cleavage with TEV protease included in the binding reaction. Protein containing assays are therefore indicated by Yhh1p-H₆ (lanes 12–20). After pre-incubation, the substrate was cleaved by addition of RNase H and oligonucleotides as indicated. For control, oligonucleotides were omitted in lanes 3 and 12. The RNA input (In) is shown in lane 2. HpaII-digested pBR322 fragments were 5′-end labelled and served as marker bands (M; lane 1). (E) S7 nuclease protection analysis. 5′-[³²P]-labelled sCYC1 RNA was pre-incubated in the absence (lane 4) and presence of increasing amounts of GST-Yhh1p-His₆ (100, 200 and 400 ng; lanes 5–7), followed by the addition of S7 nuclease. Reaction products were separated on 12% polyacrylamide/8.3 M urea gels. Also analysed were the RNA input (In; lane 1), a partial T1 nuclease digest (T1; T1 cuts RNA after guanosines) and a partial alkaline hydrolysis of the input RNA (OH^–; lane 3). To the right of the panel, the migration of full-length RNA and the position of guanosines in the RNA substrate are indicated. Arrows mark the in vitro cleavage sites and the region protected by GST-Yhh1p-His₆ is indicated.

To evaluate the strength of the Yhh1p–RNA interactions, filter binding experiments were performed. Protein was incubated with labelled CYC1 or GAL7 RNA, and RNA–protein complexes retained on nitrocellulose filters were measured by scintillation counting (see Materials and methods). These analyses revealed apparent K_D values for the interaction of Yhh1p with CYC1 and GAL7 RNAs of 5 and 15 nM, respectively (data not shown).

To characterize further the RNA-binding properties of Yhh1p, we performed RNase H protection experiments on a short CYC1 substrate (sCYC1; Figure 3C) (Dichtl and Keller, 2001). Body-labelled RNA was pre-incubated in the absence or presence of protein and subsequently supplemented with antisense DNA oligonucleotides and RNase H. Oligos 2–8 formed RNA–DNA hybrids efficiently, which targeted RNase H (Figure 3D, lanes 5–11) and formation of distinct cleavage products showed that RNase H cleavage was specifically directed by antisense oligonucleotides. Oligo 1 was not functional in this assay (lane 4). GST-Yhh1p-H₆ prevented DNA–RNA hybrid formation of oligos 6, 7 and 8 (lanes 18–20). Cleavage directed by oligo 2 was reduced in multiple experiments (lane 14). No protection could be observed when oligos 3, 4 and 5 were used (lanes 15–17). Thus, the protein bound with high affinity to sequences encompassing the poly(A) site of sCYC1. A weaker binding site resided in the efficiency element of sCYC1.

These results were corroborated by S7 nuclease protection experiments. Figure 3E shows that pre-incubation of the RNA substrate with increasing amounts of GST-Yhh1p-H₆ resulted in protection of sequences encompassing the sCYC1 poly(A) site (position 47–75) from S7 digestion. In contrast to RNase H protection experiments, this assay did not resolve binding of Yhh1p at efficiency element positions (nucleotides 19–30), possibly due to a higher stringency of the S7 protection assay.

In summary, our experiments demonstrated that Yhh1p is an RNA binding protein, which bound to CYC1 elements encompassing the poly(A) site. Since interactions observed with sequences downstream of the poly(A) site in RNase H and S7 protection experiments were not essential for binding of CYC1 RNA, as found by GST pull-down, we conclude that multiple RNA elements encompassing the CYC1 poly(A) site and possibly the poly(A) site itself are recognized. Therefore, no single sequence element was bound specifically by Yhh1p. Nevertheless, the previous observation that poly(A) site recognition by CPF (Dichtl and Keller, 2001) involves sequence elements which are also bound by Yhh1p is compatible with the idea that Yhh1p participates in poly(A) site recognition (see Discussion).

Yhh1p carries a central RNA-binding domain

The sequence of Yhh1p does not contain a known RNA-binding motif that was detectable with comparative protein analysis programs (see Materials and methods). To identify sequences required for RNA binding, N- and C-terminally truncated Yhh1p proteins were employed in GST pull-down assays. Figure 4A shows that truncation of up to 608 C-terminal residues did not affect binding of CYC1 RNA compared with full-length protein (lanes 3–6). In contrast, proteins missing >691 C-terminal residues were unable to bind (lanes 7–10). Deletion of 1016 N-terminal amino acids (aa) completely abolished RNA binding (lane 16); removal of up to 500 N-terminal residues had no effect (lanes 11–13), whereas proteins missing 583 or 663 aa, respectively, showed decreasing RNA-binding ability (lanes 14 and 15). These results indicated that a central domain within aa 500 and 750 of Yhh1p harboured the RNA-binding domain.

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Fig. 4. Delineation of the Yhh1p RNA-binding domain. (A) GST pull-down with in vitro transcribed, ³²P-labelled CYC1 RNA and 0.5 µg GST (lane 2), GST-Yhh1p-H₆ (Yhh1p; lane 3), C-terminal (lanes 4–10) and N-terminal truncations of Yhh1p (lanes 11–16) as indicated on top of the panel. Lane 1 shows 10% of RNA in binding reactions (Input). (B) Representation of internal Yhh1p domains that were expressed as N-terminal GST fusions and with a C-terminal His₆ tag. For quality control, recombinant proteins (indicated by an asterisk) were resolved by SDS–PAGE and stained with Coomassie Brilliant Blue. Migration of BSA that was included in the final elution buffer is indicated. (C) ³²P-labelled CYC1 RNA was tested for interaction with 0.5 µg GST (lane 2), GST-Yhh1p-H₆ (Yhh1p; lane 3), and Yhh1p domains as indicated in (B) (lanes 4–8). Lane 1 shows 10% of RNA in binding reactions (Input). (D) Mobility shift experiments with Yhh1p domains as indicated in (B). ³²P-labelled sCYC1 RNA was incubated with increasing amounts (200 and 400 ng) of protein and analysed by native polyacrylamide gel electrophoresis. Protein was omitted in lane 1. The position of sCYC1 is indicated.

To test this, we produced fragments that either partially or completely spanned the proposed domain (Figure 4B). In GST pull-down experiments, we found that fragments containing aa 501–666, 501–749 and 584–749 showed efficient binding of CYC1 RNA, which was comparable to full-length protein (Figure 4C, lanes 3, 4, 5 and 7). Proteins containing aa 584–666 or 664–749 (lanes 6 and 8) showed strongly reduced but detectable binding activity compared with GST (lane 2). These results were corroborated by gel mobility shift assays with the sCYC1 RNA. As shown in Figure 4D, fragments containing residues 501–666 (lanes 2 and 3), 501–749 (lanes 4 and 5) and 584–749 (lanes 8 and 9) were most efficient in formation of a slower migrating sCYC1 species. The fragment containing aa 584–666 was less efficient (lanes 6 and 7) and the fragment containing aa 664–749 was not active in this assay (lanes 10 and 11). To test whether the RNA-binding properties of the isolated RNA-binding domain were the same as with the full-length protein, we performed RNase H protection experiments with the fragment containing aa 501–749 as described in Figure 3D. The observed protection pattern paralleled that found with full-length protein (data not shown), demonstrating that the central RNA-binding domain in Yhh1p was sufficient for interaction with sCYC1.

The Yhh1p RNA-binding domain is composed of predicted β-propeller repeats

As observed by Caspary et al. (1999), Yhh1p and its homologues (CPSF 160 proteins) display sequence similarity to proteins associated with the spliceosomal U2 snRNP (SAP130) (Das et al., 1999) and to Xeroderma pigmentosum group E proteins (XPE) that are implicated in UV-damage recognition (Takao et al., 1993). A computational analysis suggested that degenerate repeat sequences within these proteins form β-propeller structures (Neuwald and Poleksic, 2000). We searched for sequences displaying homology to the Yhh1p RNA-binding domain using the NCBI BLAST server, and identified CPSF 160, SAP130 and XPE proteins. A sequence element in Yhh1p that is sufficient for RNA binding (aa 503–684) showed the most significant similarities. Figure 5 shows that the active Yhh1p RNA-binding domains, including aa 501–666 and 584–749, aligned with predicted β-propeller repeats IV–V and V–VIII, respectively (Figure 5) (Neuwald and Poleksic, 2000). These observations suggest the testable hypothesis that members of these protein families contain a nucleic acid binding motif that is related to the RNA-binding domain of Yhh1p.

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Fig. 5. Alignment of sequences related to the Yhh1p RNA-binding domain. Sequences similar to the RNA-binding domain of Yhh1p were aligned as described in Materials and methods. Invariant residues are shown in red; green indicates conserved and similar amino acid positions which are present in 70–100% of the aligned sequences, and conserved and similar residues occurring in 35–70% of the aligned sequences are shown in blue. Also indicated are sequence repeats within Yhh1p (boxes numbered I–XVI) that were predicted to form β-propeller-like structures (Neuwald and Poleksic, 2000). The sequence repeats that align with the active RNA-binding fragments of Yhh1p (aa 501–666, repeats IV–VII, and aa 584–749, repeats V–VIII) are shown in red. DDBJ/EMBL/Genbank accession numbers are: Homo Sapiens (H.s.) SAP130: CAB56791; Drosophila melanogaster (D.m.) SAP130: AAF47416; Arabidopsis thaliana (A.t.) SAP130: CAB75754; Caenorhabditis elegans (C.e.) SAP130: T32916; S.pombe (S.p.) SAP130: BAA86918; H.s. XP-E: CAA05770; A.t. XP-E: CAB81084; D.m. XP-E: AAF54901; S.p. XP-E: T37876; D.m. CPSF: AAF58240; H.s. CPSF: Q10570; C.e. CPSF: AAF36067; S.p. CPSF: T39636; S.cerevisiae (S.c.) Yhh1p: AAB64737 (YDR301w). Several more weakly related protein sequences exist in the database that were not included in this alignment.

Plasmids

Construction of plasmids used in this study is described in detail in the Supplementary data: pBD57 (YHH1-URA3-CEN), pBD64 (YHH1-LEU2-CEN), pBD75 (GST-Yhh1p-H₆), pACT–YHH1 and plasmids used for expression of N- and C-terminally truncated Yhh1p proteins and internal domains. Fusions of the CTD with GST (GST–CTD) and the GAL4 DNA binding domain (pBD–CTD) included the C-terminal 229 amino acids of Rpb1p (nucleotides 4514–5201).

Extract preparation and in vitro cleavage and polyadenylation assays

3′-end processing extracts were prepared as described previously (Ohnacker et al., 2001) Cleavage and polyadenylation assays were also carried out as described previously (Minvielle-Sebastia et al., 1994). When cleavage only was assayed, EDTA replaced MgAc and CTP replaced ATP. In a single reaction, 30–40 µg of total protein were incubated with pre-mix. For assays at elevated temperatures, pre-mix and extracts were first pre-incubated separately at 37°C for 5 min, combined and assayed at 37°C for 45 min. In vitro substrates were obtained by run-off transcription as described previously (Dichtl and Keller, 2001).

RNA analyses

Total RNAs were extracted from yeast using a hot phenol method, separated on 1.2% formaldehyde/agarose or 8.3 M urea/8% polyacrylamide gels and transferred to Hybond N⁺ (Amersham Pharmacia Biotech, Buckinghamshire, UK) membranes. Randomly primed probes were generated with [α-³²P]dATP using the Boehringer Mannheim kit. Templates were produced by polymerase chain reaction (PCR) on yeast genomic DNA covering the following sequences (numbers relative to start codon): PGK1 (1–1251), CYH2 (1–450), ADH1 (1–1049), STE3 (1–600), CYC1 (1–629), ACT1 (1–677) and ACT1-3′ (930–1536). Oligonucleotides anti-18S rRNA (CAGACAAATCACTCCA) and anti-U14 snoRNA (TCACTCAGACATCCTAGG) were labelled with [γ-³²P]ATP and T4 polynucleotide kinase. RNase H reactions on total RNA were carried out with oligo ACT1-RNaseH (CTGGGAACA TGGTGGTACCACC) and RNAs were separated on 8.3 M urea/6% polyacrylamide gels. Poly(A)-tail analysis was performed as described previously (Minvielle-Sebastia et al., 1998). Transcriptional run-on analysis was performed according to Birse et al. (1998).

Recombinant protein expression and purification

Escherichia coli BL21 cells transformed with the respective plasmids were grown in 2× YT at 25°C to an OD₆₀₀ of 2.0. After induction with 0.5 mM IPTG, incubation was continued for 6 h. Proteins carrying a C-terminal His₆ tag were first purified with Ni²⁺-NTA (Qiagen) according to the manufacturer’s instructions. GST fusion proteins and double-tagged constructs were purified on glutathione–Sepharose 4B as recommended (Amersham Pharmacia Biotech, Piscataway, NJ); however, all steps were performed at 4°C or on ice. Proteins were eluted with buffer G [10 mM reduced glutathione, 50 mM Tris–HCl pH 8.0, 0.01% NP-40, 0.01 mg/ml bovine serum albumin (BSA), 10% glycerol].

Protein–protein interactions

Phosphorylation of GST–CTD was performed as described previously (Hirose and Manley, 1998). GST, GST–CTD and phosphorylated GST–CTD proteins were bound to 100 µl glutathione–Sepharose 4B (Pharmacia) at 0.02 mg/ml in buffer P [phosphate-buffered saline containing 0.02% NP-40, 1 mM dithiothreitol (DTT), 0.4 µg/ml leupeptin, 0.7 µg/ml pepstatin] for 1 h at 4°C. Beads were washed five times with buffer P containing 1 M NaCl, equilibrated twice with buffer P, and resuspended in 1 ml buffer P supplemented with 0.1 mg/ml BSA. Phosphorylation of the CTD was controlled by immunodetection with an antibody directed against phosphoserine 5 (H14, BAbCO, Richmond, CA) or the unphosphorylated CTD of pol II (8WG16, BAbCO). In vitro translations were performed with the TNT-coupled transcription– translation system (Promega). For GST pull-down, 45 µl GST or GST-fusion protein were incubated with in vitro translated ³⁵S-labelled proteins for 1.5 h at 4°C. The resin was washed four times with 1 ml buffer P for 20 min at 4°C. Proteins were eluted in sample buffer and resolved by SDS–PAGE.

RNA–protein interactions

Pull-down experiments with GST fusion proteins and CYC1 and GAL7 wild-type and mutant RNAs, and RNase H protection experiments were performed as described previously (Dichtl and Keller, 2001). Filter-binding experiments were performed in a 50-µl volume in RNA binding buffer (RBB) [13 mM HEPES–KOH pH 7.9, 28 mM (NH₄)₂SO₄, 33 mM KCl, 1 mM MgCl₂, 1 mM EDTA, 0.2 mM DTT, 0.01% NP-40]. Internally labelled CYC1 or GAL7 RNA (20 fmol) were incubated with 0, 5, 10, 100, 400, 800 and 1500 fmol GST-Yhh1p-H₆ for 15 min at room temperature. The reactions were applied to NC20 nitrocellulose filters (Schleicher and Schuell, Dassel, Germany) that were pre-treated with RBB containing 10 µg E.coli tRNA. Following a wash step with 1.5 ml RBB, the radioactivity retained on the filters was determined by scintillation counting with Emulsifier Safe liquid (Packard, Groningen, Netherlands). Apparent K_D values were determined as described by Wahle et al. (1993). S7 nuclease protection experiments, partial alkaline hydrolysis and nuclease T1 digest were performed as described previously (Knapp, 1989) with modifications. One hundred femtomoles of 5′-[³²P]-labelled sCYC1 RNA was pre-incubated in the absence and presence of 100, 200 and 400 ng of GST-Yhh1p-His₆ in buffer (25 mM Tris–HCl pH 8.0, 30 mM KAc, 5 mM MgAc, 1 mM DTT) for 10 min at 37°C. Then, 0.1 U of S7 nuclease was added and reactions were stopped after 6 min by the addition of loading buffer containing 100 mM EDTA. Reaction products were separated on 12% polyacrylamide/8.3 M urea gels. Electromobility shift assays were performed with proteins as indicated and 20 fmol sCYC1 RNA in 10 µl containing 100 mM KCl, 1 mM MgCl₂, 1 mM DTT, 0.5 U RNA Guard and 100 ng E.coli tRNA. Following pre-incubation at 30°C for 15 min, reactions were separated on native 8% polyacrylamide gels (acrylamide/bisacrylamide 80:1 in 25 mM Tris base, 25 mM boric acid, 1 mM EDTA) at room temperature for 4 h at 200 V.

Computing

Protein and nucleotide sequence searches were performed with the BLASTP and TBLASTX programs on the NCBI server (Altschul et al., 1997) (http://www.ncbi.nlm.nih.gov). The Yhh1p RNA-binding domain and corresponding similar sequences were aligned with the CLUSTAL_X program (Thompson et al., 1997) and subsequent manual refinement.