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J. Biol. Chem., Vol. 276, Issue 30, 28075-28082, July 27, 2001

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The Length, Phosphorylation State, and Primary Structure of the RNA Polymerase II Carboxyl-terminal Domain Dictate Interactions with mRNA Capping Enzymes^*

Yi Pei, Stéphane Hausmann, C. Kiong Ho, Beate Schwer^§, and Stewart Shuman^¶

From the  Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021 and the ^§ Microbiology and Immunology Department, Weill Medical College of Cornell University, New York, New York 10021

Received for publication, March 9, 2001, and in revised form, May 11, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The carboxyl-terminal domain (CTD) of elongating RNA polymerase II serves as a landing pad for macromolecular assemblies that regulate mRNA synthesis and processing. The capping apparatus is the first of the assemblies to act on the nascent pre-mRNA and the one for which binding of the catalytic components is most clearly dependent on CTD phosphorylation. The present study highlights a distinctive strategy of cap targeting in fission yeast whereby the triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes of the capping apparatus do not interact physically with each other (as they do in budding yeast and metazoans), but instead bind independently to the phosphorylated CTD. In vivo interactions of Pct1 and Pce1 with the CTD in a two-hybrid assay require 12 and 14 tandem repeats of the CTD heptapeptide, respectively. Pct1 and Pce1 bind in vitro to synthetic CTD peptides containing phosphoserine uniquely at position 5 or doubly at positions 2 and 5 of each of four tandem YSPTSPS repeats, but they bind weakly (Pce1) or not at all (Pct1) to a peptide containing phosphoserine at position 2. These results illustrate how remodeling of the CTD phosphorylation array might influence the recruitment and dissociation of the capping enzymes during elongation. But how does the CTD structure itself dictate interactions with the RNA processing enzymes independent of the phosphorylation state? Using CTD-Ser5 phosphopeptides containing alanine substitutions at other positions of the heptad, we define essential roles for Tyr-1 and Pro-3 (but not Thr-4 or Pro-6) in the binding of Schizosaccharomyces pombe guanylyltransferase. Tyr-1 is also essential for binding and allosteric activation of mammalian guanylyltransferase by CTD Ser5-PO₄, whereas alanine mutations of Pro-3 and Pro-6 reduce the affinity for the allosteric CTD-binding site. These are the first structure-activity relationships deduced for an effector function of the phosphorylated CTD.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

mRNA capping occurs co-transcriptionally by a series of three enzymatic reactions in which the 5'-triphosphate terminus of the pre-mRNA is cleaved to a diphosphate by RNA triphosphatase, then capped with GMP by RNA guanylyltransferase, and methylated at the N-7 position of guanine by RNA (guanine-7) methyltransferase (1). Specific targeting of cap formation to transcripts made by RNA polymerase II (pol II)¹ is achieved, at least in part, through physical interactions of one or more components of the capping apparatus with the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of pol II (2-5). The CTD, which is unique to pol II, is composed of a tandemly repeated heptad motif (consensus sequence = YSPTSPS). The mammalian pol II large subunit has 52 heptad repeats, the fission yeast Schizosaccharomyces pombe subunit has 29 repeats, and the budding yeast Saccharomyces cerevisiae subunit has 27 copies (6). The CTD undergoes a cycle of extensive phosphorylation and dephosphorylation of Ser-5 and Ser-2, which is coordinated with the transcription cycle.

In the budding yeast S. cerevisiae, the guanylyltransferase (Ceg1) and methyltransferase (Abd1) components of the capping apparatus bind independently in vitro and in vivo to the phosphorylated CTD, but not to unphosphorylated CTD (2, 7). The S. cerevisiae RNA triphosphatase Cet1 does not bind to the CTD by itself (8), but it does bind to Ceg1 (9-11) and may thus be escorted to the transcription complex together with Ceg1. The mammalian capping enzyme Mce1 is a bifunctional polypeptide composed of an NH₂-terminal RNA triphosphatase domain linked to a COOH-terminal guanylyltransferase domain. Mce1 binds to the pol II CTD and this interaction also requires CTD phosphorylation (2, 5). The guanylyltransferase domain Mce1(211-597) per se binds to CTD-PO₄, but the NH₂-terminal triphosphatase domain Mce1(1-210) does not (3, 12). Apparently, the covalent connection between the mammalian guanylyltransferase and triphosphatase domains allows the guanylyltransferase to deliver the triphosphatase to the transcription elongation complex. The mammalian guanylyltransferase can even deliver heterologous viral or fungal RNA triphosphatases in vivo when they are fused to create chimeric capping enzymes (13).

Recent studies indicate that the CTD and CTD phosphorylation play more than mere architectural roles in coordinating mRNA synthesis and processing (reviewed in Ref. 14). For example, the phosphorylated CTD directly stimulates the catalytic activity of the mammalian RNA guanylyltransferase (12, 15). The remarkable feature of the activation of mammalian guanylyltransferase by the CTD is the requirement for phosphoserine at position 5. The guanylyltransferase also binds to CTD phosphorylated on Ser-2, but this interaction has no effect on enzyme activity (12).

Deletion analyses of the pol II large subunit have shown that the growth of fungal and metazoan cells is contingent on a minimal CTD length. Mammalian cells cannot grow with 25 or fewer CTD heptad repeats, but are fully viable with 36 repeats (16). West and Corden (17) found that truncating the S. cerevisiae CTD to 7 heptad repeats was lethal, whereas deletions leaving 8 or 9 heptads elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects, and normal cell growth required at least 10 integral copies of the heptad. A slightly higher threshold for yeast CTD length was reported by Nonet et al. (18), whereby less than 10 heptad repeat was lethal, 10 to 12 repeats resulted in conditional phenotypes, and 13 or more repeats sustained normal growth. Mutations that eliminate the hydroxyl moieties (potential phosphate acceptors) on Tyr-1, Ser-2, or Ser-5 of the heptad sequence are lethal to S. cerevisiae (17), but it is not clear if the loss of CTD function in vivo is a direct consequence of a perturbation of CTD structure (e.g. intra- or intermolecular loss of hydrogen bonding interactions) or a secondary effect on CTD phosphorylation state.

There is relatively little known about the fine structure of the CTD and how such parameters as CTD length, amino acid sequence, and phosphorylation arrays influence various CTD-PO₄ effector functions. The interaction of the capping apparatus with the phosphorylated CTD provides an attractive model system to address these issues. Here we present an analysis of the interactions of the capping enzymes of the fission yeast S. pombe with the CTD of the largest subunit of S. pombe pol II that highlights a new strategy for independent recruitment of separately encoded triphosphatase (Pct1) and guanylyltransferase (Pce1) components to the pol II elongation complex. Analysis of the interaction of S. pombe and mammalian capping enzymes with mutated synthetic CTD phosphopeptides illuminates for the first time the contributions of conserved CTD side chains to CTD function independent of any effects on CTD phosphorylation. This approach should be applicable to the study of CTD-PO₄ effector roles in other co-transcriptional RNA transactions such as pre-mRNA splicing, polyadenylation, and transcription termination.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Yeast Two-hybrid Screen-- The screen was performed using the Matchmaker system from CLONTECH with protocols specified by the vendor. The full-length PCT1 and PCE1 genes were inserted into pAS2-1 (2µ TRP1) so as to fuse them in-frame with the GAL4 DNA-binding domain (BD). The BD-PCT1 and BD-PCE1 plasmids were transformed into S. cerevisiae strain Y190. Trp⁺ isolates were selected and grown in 1-liter cultures SD(Trp) medium. The cells were harvested and transformed by the LiCl method with an S. pombe cDNA library in a GAL4 activation domain (AD) fusion plasmid (2µ LEU2) (19). The transformant pool was selected directly for HIS3 reporter expression by plating on SD(-Trp, -Leu, -His) agar containing 25 mM 3-amino-1,2,4-triazole. The total number of transformants screened was estimated by plating an aliquot of the pool on SD(-Trp, -Leu) agar. The His⁺ colonies were selected after incubation for 3 to 7 days at 30 °C, then patched and restreaked on SD(-Trp, -Leu, -His, 3-amino-1,2,4-triazole) agar. Single colonies that retested for His⁺ were patched and tested for lacZ reporter expression using the -galactosidase colony-lift filter assay. DNA recovered from the strains that tested positive for both HIS3 and lacZ expression was used as the template for PCR amplification of the S. pombe DNA insert with flanking primers specific for the AD-fusion plasmid. The PCR products were gel-purified and then sequenced. The AD plasmid clones were recovered after transformation into Escherichia coli DH5.

CTD Truncation Mutants-- Gene fragments encoding serially truncated versions of S. pombe Rpb1 were generated by PCR amplification using antisense primers that introduced stop codons in lieu of the codons for amino acids 1687, 1669, 1648, 1634, 1592, or 1585 and a XhoI site immediately 3' of the stop codon. The sense primer introduced a BamHI site at the codon for amino acid 1516. The PCR products were digested with BamHI and XhoI and then inserted into the two-hybrid AD fusion vector pGAD-GH. Sequencing of the inserts in the resulting series of AD-Rbp1 plasmids verified that the truncated RPB1 genes were fused in-frame to AD and that no unwanted coding changes had been introduced during amplification and cloning.

Capping Enzymes-- S. pombe RNA triphosphatase Pct1 and mouse guanylyltransferase Mce1(211-597) were produced in E. coli as N-terminal His-tagged fusions and purified from soluble bacterial lysates by nickel-agarose chromatography as described previously (3, 20). The Mce1(211-597)-[³²P]GMP intermediate was prepared by reaction of 20 µg of Mce1(211-597) with 5 µM [-³²P]GTP and 2.5 mM MgCl₂ for 30 min at 30 °C. The reaction mixture was adjusted to 25 mM EDTA and 10% glycerol and the native enzyme-GMP intermediate resolved from free [³²P]GTP by gel filtration through a 1-ml column of Sephadex G-50.

S. pombe RNA guanylyltransferase Pce1 was produced in E. coli as a His-tagged fusion as follows. The pET-PCE1 plasmid (21) was modified by insertion of a DNA fragment encoding an NH₂-terminal 21-amino acid peptide (MGHHHHHHHHHHSSGHIEGRP) in-frame with the open reading frame encoding the 402-amino acid Pce1 polypeptide. The pETHis-PCE1 plasmid was transformed into E. coli BL21(DE3). A 250-ml culture was grown at 37 °C in LB medium containing 0.1 mg/ml ampicillin until the A₆₀₀ reached 0.5. The culture was adjusted to 0.4 mM isopropyl-1-thio--D-galactopyranoside and 2% ethanol and incubation was continued for 20 h at 17 °C. Cells were harvested by centrifugation and stored at 80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria were resuspended in 25 ml of buffer A (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10% glycerol). Phenylmethylsulfonyl fluoride and lysozyme were added to final concentrations of 300 µM and 100 µg/ml, respectively. After incubation on ice for 30 min, Triton X-100 was added to a final concentration of 0.1% and the lysate was sonicated to reduce viscosity. Insoluble material was removed by centrifugation for 45 min at 18,000 rpm in a Sorvall SS34 rotor. The soluble extract was mixed for 30 min with 1 ml of Ni²⁺-NTA-agarose (Qiagen) that had been equilibrated with buffer A containing 0.1% Triton X-100 and 25 mM imidazole. The slurry was poured into a column and the resin was washed with 10 ml of buffer A containing 0.1% Triton X-100 and 50 mM imidazole. Pce1 was then step-eluted with 250 mM imidazole in buffer A. The enzyme preparation (7 mg of protein) was dialyzed against buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM dithiothreitol, 5% glycerol, 0.03% Triton X-100, and stored at 80 °C. The protein concentration was determined using the Bio-Rad dye-binding method with bovine serum albumin as the standard.

CTD Phosphopeptides-- NH₂-terminal biotinylated CTD phosphopeptides composed of 4 tandem YSPTSPS repeats containing phosphoserine at position 2, position 5, or positions 2 plus 5 of each repeat were synthesized and purified as described previously (12, 22). Mutant Ser-5 phosphopeptides Y1A (ASPTSPS)₄, P3A (YSATSPS)₄, T4A (YSPASPS)₄, and P6A (YSPSTAS)₄ were synthesized and purified using the same methods. The molecular weights of the mutant phosphopeptides were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The measured masses were in agreement with the calculated theoretical masses within the limits of calibration of the instrument. The peptides were dissolved in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA and stored at 4 °C.

The biotinylated CTD peptides were absorbed to streptavidin-coated magnetic beads (Dynabeads M280 streptavidin; Dynal) in binding buffer (25 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol, 5% glycerol, 0.03% Triton X-100) containing 50 mM NaCl as described previously (12). The amount of biotinylated CTD peptide was sufficient to saturate the bead-bound streptavidin. After peptide adsorption, the beads were washed three times with binding buffer to remove any unbound peptide.

CTD Affinity Chromatography-- Affinity chromatography was performed by mixing 14 µg (280 pmol) of Pce1, 9 µg (240 pmol) of Pct1, or 4 µg (85 pmol) of Mce1(211-597) with 0.6 mg of CTD peptide beads (estimated to contain 390 pmol of peptide) in 50 µl of binding buffer with 50 mM NaCl. After incubation for 30 min on ice, the beads were concentrated by microcentrifugation for 15 s and then held in place with a magnet as the supernatant was withdrawn. The beads were resuspended in 1 ml of binding buffer and subjected to two cycles of concentration and washing. After the third wash, the beads were resuspended in 50 µl of binding buffer. Aliquots (20 µl) of the bead bound fraction were mixed with 4 µl of SDS sample buffer (200 mM Tris-HCl, pH 6.8, 8% SDS, 140 nM -mercaptoethanol, 40% glycerol), heated at 90 °C for 3-5 min, and then analyzed by SDS-PAGE.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

S. pombe RNA Triphosphatase and RNA Guanylyltransferase do Not Interact Physically-- The triphosphatase and guanylyltransferase components of fungal capping systems have conserved catalytic domains (1), but the enzymes are not always functionally interchangeable in vivo. S. pombe and Candida albicans guanylyltransferases can function in S. cerevisiae with the endogenous Cet1 triphosphatase (21, 23), but the ability of heterologous triphosphatases to function with the S. cerevisiae guanylyltransferase is variable and correlates with the presence or absence of a conserved guanylyltransferase-binding domain on the surface of the heterologous RNA triphosphatase (11, 24). S. pombe triphosphatase Pct1 (a 303-amino acid polypeptide), which lacks the surface domain and does not function on its own in S. cerevisiae, can support cell growth when S. pombe guanylyltransferase Pce1 (a 402-amino acid polypeptide) is provided in trans (20). Thus, the fission yeast capping components display a species-specific genetic interaction.

Given that the triphosphatase and guanylyltransferase components of the capping apparatus are physically associated in budding yeast and metazoans (with the interaction being noncovalent in S. cerevisiae and covalent in metazoans), the simplest model to account for the genetic interaction would be that S. pombe triphosphatase Pct1 forms a complex with S. pombe guanylyltransferase Pce1 and that Pce1, which binds in vitro to CTD-PO₄ (2), in turn chaperones the triphosphatase to the transcription complex. To test this hypothesis, we asked whether a mixture of recombinant Pct1 (a homodimer) and recombinant Pce1 (a monomeric protein) resulted in the formation of a stable heteromeric complex that could be isolated by velocity sedimentation using the methods that readily detect formation of a complex between S. cerevisiae Cet1 and Ceg1 (10). We were unable to detect any complex formation between the S. pombe Pct1 and Pce1 (not shown). Next, we conducted a two-hybrid screen of a S. pombe cDNA library linked to the Gal4 activation domain (AD) using as "bait" either Pce1 or Pct1 fused to the Gal4 DNA-BD. Although the screens yielded multiple interacting clones in each case (see below), we did not isolate Pce1 or a fragment thereof using Pct1 as bait. Nor did we isolate Pct1 or a fragment thereof using Pce1 as bait. To exclude the possibility of a false negative caused by absence of the Pce1 or Pct1 genes from the S. pombe plasmid library, we performed a directed two-hybrid interaction assay using an explicitly engineered pair of BD-Pce1 and AD-Pct1 vectors. Here again, we saw no evidence of interaction between the S. pombe triphosphatase and guanylyltransferase (not shown). Note that the S. cerevisiae and C. albicans triphosphatase and guanylyltransferase components display two-hybrid interactions with one another (25).

S. pombe Triphosphatase and Guanylyltransferase Interact with S. pombe CTD in Vivo-- A two-hybrid screen of ~100,000 transformants for triphosphatase-interacting proteins using BD-Pct1 as bait yielded 16 His⁺ isolates, of which 8 contained plasmids encoding AD fused in-frame to a COOH-terminal fragment of Rpb1, the largest subunit of S. pombe pol II (26). Three different AD-Rpb1 fusion clones were isolated: AD-Rpb1(1305-1752) was recovered twice; AD-Rpb1(1324-1752) was recovered 5 times; and AD-Rpb1(1478-1752) was isolated once (Fig. 1). Control experiments showed that the His⁺ and lacZ⁺ phenotypes required co-transformation with BD-Pct1 and AD-Rpb1(1305-1752) plasmids and that neither fusion plasmid was able to drive expression of the HIS3 or lacZ reporter genes when co-transformed with the BD or AD vectors (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   S. pombe capping enzymes interact with the Rpb1 CTD in vivo. Plasmids encoding the indicated AD-Rpb1 fusions were transformed into S. cerevisiae Y190 cells bearing the BD-PCE1 or BD-PCT1 plasmid. The limits of the Rpb1 polypeptide segment of the fusion protein are indicated and drawn to scale as horizontal lines. The CTD heptad repeats are depicted as vertical bars. The AD-Rpb1 fusions that were recovered in the two-hybrid screen of the S. pombe cDNA library are indicated by check marks. Other derivatives were engineered by PCR-based site-directed mutagenesis. Single Trp+ Leu+ isolates were selected and streaked on SD(-Trp, -Leu, -His, 3-amino-1,2,4-triazole) agar medium to test for HIS3 expression. Robust growth on selective medium was scored as ++ (see Fig. 2A). Strains that grew no better than the DB-PCT1 plus AD control (see Fig. 2A) were scored as minus (). Intermediate levels of growth were scored as + or ± on the basis of His+ colony size.

View larger version (53K): [in this window] [in a new window]   Fig. 2.   Specificity of the Pct1-CTD two-hybrid interaction. The BD-Pct1 and AD-Rpb1(1305-1752) fusion plasmids and the BD and AD vectors without inserts were transformed pairwise into S. cerevisiae Y190. Panel A, single Trp+ Leu+ isolates containing the indicated plasmid pairs were selected and streaked on SD(-Trp, -Leu, -His, 3-amino-1,2,4-triazole) agar medium. The plate was photographed after incubation for 5 days at 30 °C. Panel B, Trp+ Leu+ isolates containing the indicated plasmid pairs were patched on SD(-Trp, -Leu) agar medium. The cell patches were photographed after incubation for 1 day at 30 °C (Growth). The cell patches were then tested for -galactosidase activity using the colony-lift filter assay. A photograph of the filter is shown (lacZ).

A two-hybrid screen of ~200,000 transformants for guanylyltransferase-interacting proteins using BD-Pce1 as bait yielded 11 His⁺ isolates. Sequencing of the AD plasmids from these strains showed that 8 of the isolates encoded AD fused in-frame to a COOH-terminal fragment of Rpb1 from residues 1324 to 1752 (Fig. 1). Control transformations confirmed that HIS3 and lacZ expression required co-transformation with the BD-Pce1 and AD-Rpb1(1324-1752) plasmids (not shown).

Minimal CTD Length Required for Interaction with S. pombe Capping Enzymes in Vivo-- The AD-Rpb1 fusions that interacted in vivo with Pce1 or Pct1 contained all 29 tandem copies of the heptad repeat plus a variable segment of the pol II subunit upstream of the start of the CTD at position 1551 (26). To gauge the role of the non-reiterated protein segment, we constructed an AD-Rpb1(1516-1752) fusion clone and tested it in a directed two-hybrid assay paired with BD-Pce1 and BD-Pct1. We found that the Rpb1 interaction with both capping enzymes persisted when the AD fusion contained little more than the CTD repeats per se. Thus, subsequent COOH-terminal truncations of the CTD were performed with the NH₂-terminal position of Rpb1 fixed at 1516 (Fig. 1).

The salient findings were that the CTD interaction with Pct1 persisted with undiminished strength (as gauged by colony size during selection for His⁺) with 19, 17, or 14 heptad repeats, with diminished strength after truncation to 12 heptad repeats, and was eliminated with 6 or fewer heptads (Fig. 1). The two-hybrid CTD interaction with Pce1 displayed a more stringent requirement for CTD length; the interaction was of diminished strength at between 19 and 14 heptad repeats and was abolished at 12 or fewer repeats (Fig. 1).

Binding of S. pombe Capping Enzymes to Defined Synthetic CTD Phosphopeptides-- Previous CTD-binding studies in vitro involving S. pombe guanylyltransferase employed an immobilized ligand composed of recombinant GST-CTD that was enzymatically phosphorylated in vitro using HeLa cell extract as the source of CTD kinase. The fusion protein contained 15 copies of the YSPTSPS heptad sequence and an average of 3 serine-phosphates per GST-CTD polypeptide (2). We have since developed synthetic CTD phosphopeptide ligands, in which the numbers and positions of the phosphates are known and subject to manipulation, to delineate the requirements for the interaction of capping enzymes with the CTD (12). Here we employed 28-mer CTD peptide ligands composed of four tandem repeats of the YSPTSPS heptad that contained phosphoserine at either position 2 or 5 of each heptad or phosphoserine at both positions 2 and 5 of each heptad. An NH₂-terminal biotin moiety was added during chemical synthesis so that the peptides could be linked to streptavidin-coated beads for affinity chromatography purposes. The CTD phosphopeptide-containing beads, or control beads containing an unphosphorylated 28-mer CTD peptide, were incubated with purified recombinant Pce1. The beads were recovered by centrifugation and washed three times with buffer. The bead-bound material was eluted with 1% SDS. The input guanylyltransferase protein (L) and the bead-bound fractions were then analyzed by SDS-PAGE. The key findings were that the S. pombe guanylyltransferase bound equally well to the CTD Ser5-PO₄ peptide and the Ser2-PO₄/Ser5-PO₄ peptide. Slightly less than one-fifth of the input protein was retained on the beads in both cases. Pce1 bound less avidly to the CTD Ser2-PO₄ peptide. The binding was specific for CTD-PO₄, because Pce1 did not bind at all to the beads containing the unphosphorylated CTD peptide (Fig. 3A). Potential effects of the CTD Ser2-PO₄/Ser5-PO₄ peptide on Pce1 activity were gauged by preincubating recombinant Pce1 with increasing amounts of the peptide (up to 120-fold molar excess of peptide over Pce1) and then assaying the mixtures for enzyme-GMP complex formation during a reaction in vitro with [-³²P]GTP. The phosphopeptide had no significant stimulatory or inhibitory effect on Pce1 activity (not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Binding of S. pombe capping enzymes to the phosphorylated CTD. A, the CTD peptide with 4 tandem heptad repeats is shown. An NH2-terminal biotin anchors the peptide to a streptavidin-coated magnetic bead. Aliquots of Pce1 (14 µg) (panel A) and Pct1 (9 µg) (panel B) were subjected to CTD affinity chromatography using beads with immobilized CTD peptides: either unphosphorylated () or phosphorylated at Ser-2, Ser-5, or Ser-2 + Ser-5 of each heptad repeat. Aliquots comprising 8% of the input Pce1 or 4% of the input Pct1 (L) and 40% of the CTD-bound SDS eluate fractions were analyzed by SDS-PAGE and the polypeptides were visualized by staining with Coomassie Blue dye.

The novel finding was that the recombinant S. pombe RNA triphosphatase by itself bound specifically to CTD peptides phosphorylated on Ser-5 and not to the unphosphorylated peptide or the Ser2-PO₄ peptide (Fig. 3B). Pct1 bound more avidly to the doubly phosphorylated Ser2-PO₄/Ser5-PO₄ peptide (~5% of input protein retained on the bead) compared with the Ser5-PO₄ peptide.

These experiments show that: (i) S. pombe guanylyltransferase and RNA triphosphatase bind independently to CTD-PO₄; (ii) four heptad repeats suffice for binding; and (iii) the affinities of Pce1 and Pct1 for the CTD are affected by the site of serine phosphorylation (2 versus 5) or the complexity of the phosphorylation array (5 versus 2 plus 5). Thus, the S. pombe capping system is so far unique in that the triphosphatase component has an intrinsic capacity to recognize the CTD-PO₄ independent of the guanylyltransferase.

Role of CTD-PO₄ Primary Structure in Binding of S. pombe Capping Enzymes-- Pct1 and Pce1 both require serine phosphorylation to bind to the CTD in vitro; hence, we surmise that the true interacting partner in the in vivo two-hybrid assay is an AD-Rpb1 fusion protein that has been phosphorylated by one or more of the CTD kinases resident in S. cerevisiae. Thus, it is possible that the observed requirement for at least 12 or 14 heptad repeats for a two-hybrid interaction of the CTD with Pct1 or Pce1 in vivo (these values being much greater than the 4 repeats sufficient for detection of the interactions in vitro) is reflective of the CTD length requirements for attaining the proper phosphorylation array(s) to which the capping enzymes can bind. This scenario highlights the inherent limitations to structure-function analyses of phosphorylation-dependent CTD interactions in vivo: (i) the true structure of the CTD-PO₄ ligand in the cell is not readily ascertained; (ii) effects of CTD mutations on the biological readout can be caused indirectly by affecting CTD phosphorylation by cyclin-dependent protein kinases or dephosphorylation by CTD phosphatases; and (iii) it is difficult to distinguish effects on phosphorylation dynamics from phosphorylation-independent alterations in CTD structure that influence ligand binding.

To circumvent these limitations and establish clear structure-activity relationships for CTD binding to the S. pombe guanylyltransferase, we used mutated versions of the synthetic 28-mer CTD Ser5-PO₄ peptides in which Tyr-1, Pro-3, Thr-4, or Pro-6 were replaced by alanine in each of the four tandem heptad repeats. The Y1A and P3A mutations abolished Pce1 binding to the CTD Ser5-PO₄ peptide immobilized on streptavidin beads, whereas the T4A and P6A mutations were without significant effect (Fig. 4A). Thus, Tyr-1 and Pro-3 are critical for Pce1 binding independent of any possible effects on phosphorylation, which is not a variable in this experiment. The lack of a requirement for the Pro-6 side chain for Pce1 binding to phosphorylated CTD is an instructive finding that would be difficult to establish without using a chemically synthesized Ser5-PO₄ CTD ligand, because the conserved proline at position 6 is a component of the Ser-Pro specificity determinant for the cyclin-dependent kinases that catalyze the enzymatic phosphorylation of the CTD on the neighboring Ser-5.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Effects of alanine mutations on binding of the S. pombe guanylyltransferase to CTD Ser5-PO4. The wild-type (WT) CTD peptide with 4 tandem heptad repeats containing phosphoserine at position 5 is shown. An NH2-terminal biotin anchors the peptide to a streptavidin-coated magnetic bead. Aliquots of Pce1 (14 µg) (panel A) or Pct1 (15 µg) (panel B) were subjected to affinity chromatography using beads with immobilized WT CTD Ser5-PO4 peptide or the indicated mutant peptide. Aliquots comprising 40% of the bead-bound fraction were analyzed by SDS-PAGE and the polypeptides were visualized by staining with Coomassie Blue dye.

An analysis of the binding of S. pombe triphosphatase to the WT and mutant 28-mer CTD Ser5-PO₄ peptides is shown in Fig. 4B. The Y1A, P3A, and P6A mutations abolished Pct1 binding, whereas the T4A mutation had no adverse effect.

Role of CTD Structure in Binding and Activation of Mammalian Guanylyltransferase-- We showed previously that phosphorylation of either Ser-2 or Ser-5 within the synthetic (YSPTSPS)₄ ligand was sufficient for the CTD to bind the guanylyltransferase domain of mammalian capping enzyme, but that stimulation of guanylyltransferase activity was a unique property of CTD Ser5-PO₄ (12). Moreover, because stimulation of enzyme-GMP complex formation by the CTD Ser5-PO₄ peptide was undiminished by an excess of the CTD Ser2-PO₄ peptide, it was proposed that the mammalian capping enzyme has two binding sites for CTD-PO₄: an allosteric activator site that is specific for the Ser-5 phosphopeptide and a second binding site that accepts the Ser-2 phosphopeptide. This model raises the question of whether phosphorylation of both Ser-2 and Ser-5 would alter the interactions of the CTD with mammalian guanylyltransferase.

We compared the binding of Mce1(211-597) to a series of 28-mer synthetic CTD peptides containing either Ser5-PO₄, Ser2-PO₄ plus Ser5-PO₄, or no phosphates. To facilitate quantitation of guanylyltransferase binding to the immobilized peptides, we mixed the recombinant Mce1(211-597) protein with an aliquot of the covalent Mce1(211-597)-[³²P]GMP intermediate that had been isolated by gel filtration. The radiolabeled species comprised ~5% of protein applied to the CTD peptide beads. Analysis of the total guanylyltransferase by SDS-PAGE and Coomassie Blue staining indicated that the enzyme bound better to the Ser2-PO₄/Ser5-PO₄ peptide than to the Ser5-PO₄ peptide and there was only trace binding to the unphosphorylated CTD peptide (Fig. 5A). Analysis of the bound Mce1(211-597)-[³²P]GMP intermediate by SDS-PAGE and scanning with a PhosphorImager confirmed these results (Fig. 5B).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effects of alanine mutations on binding of the mammalian guanylyltransferase to CTD Ser5-PO4. Aliquots of the mammalian guanylyltransferase (a mixture of 4 µg of the recombinant Mce1(211-597) protein plus 0.2 µg of the isolated Mce1(211-597)-[32P]GMP intermediate) were subjected to affinity chromatography using beads with immobilized CTD peptides consisting of 4 heptad repeats: either unphosphorylated (CTD), phosphorylated at Ser-5 (CTD Ser5-PO4), or phosphorylated at Ser-2 plus Ser-5 (2 + 5). Aliquots comprising 40% of the input enzyme (L) and 40% of the bead-bound SDS eluate fraction were analyzed by SDS-PAGE and the Mce1(211-597) polypeptides were visualized by staining with Coomassie Blue dye (panel A). Aliquots comprising 10% of the bead-bound SDS eluate fraction were resolved by SDS-PAGE and the signal intensities (PSL) of the bound Mce1(211-597)-[32P]GMP complexes were quantitated by scanning the gel with a PhosphorImager (panel B).

In a separate experiment, we tested the effects of the CTD phosphopeptides on guanylyltransferase activity (Fig. 6). Mce1(211-597) was preincubated on ice with increasing concentrations of the Ser2-PO₄/Ser5-PO₄ or Ser5-PO₄ peptides and aliquots of the mixtures were then assayed for enzyme-GMP complex formation. We found that formation of the enzyme-GMP complex was stimulated 2.5-fold by either CTD phosphopeptide. The dependence of the extent of stimulation on peptide concentration was similar for the Ser2-PO₄/Ser5-PO₄ and Ser5-PO₄ ligands. Stimulation was maximal at 2.5 µM peptide (Fig. 6) and increasing the peptide concentration to 5 µM elicited no further stimulation (not shown). Half-maximal stimulation was attained at peptide concentrations of 0.2 to 0.3 µM (with guanylyltransferase concentration being 0.1 µM). We conclude that the guanylyltransferase activation function of Ser-5 phosphorylation is "dominant" over the non-activating Ser-2 phosphorylation and that the putative allosteric site on the mammalian guanylyltransferase does not discriminate significantly between the Ser-2/Ser-5 and Ser-5 phosphorylation arrays.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effects of alanine mutations on stimulation of the guanylyltransferase activity of mammalian capping enzyme by CTD Ser5-PO4. Mce1(211-597) (20 pmol) was preincubated for 15 min on ice with 0, 63, 125, 250, 500, or 1000 pmol of the indicated CTD phosphopeptides in 10 µl of binding buffer containing 25 mM NaCl. An aliquot (1 µl) of each mixture was then assayed for enzyme-GMP complex formation in a reaction mixture (20 µl) containing 50 mM Tris-HCl (pH 8.0), 2 mM dithiothreitol, 5 mM MgCl2, and 0.17 µM [-32P]GTP. The reactions were quenched with SDS after incubation for 10 min at 37 °C and the products were analyzed by SDS-PAGE. The signal intensities of the Mce1(211-597)-[32P]GMP complexes were quantitated by scanning the dried gel with a PhosphorImager and then normalized to that of the no CTD control reaction (defined as 1.0). The fold stimulation is plotted as a function of the final concentration of peptide in the guanylyltransferase reaction, which contained 0.1 µM Mce1(211-597). The data shown are averages of three independent experiments.

The effects of position-specific alanine substitutions within the four heptad repeats of the CTD Ser5-PO₄ peptide on binding to the mammalian guanylyltransferase are shown in Fig. 5. Whereas changing Thr-4 to alanine had no effect, the introduction of alanine in lieu of Tyr-1 virtually abolished the affinity of CTD Ser5-PO₄ for the guanylyltransferase. Changing either Pro-3 or Pro-6 to alanine reduced the extents of capping enzyme binding to about one-third of that seen with the wild type CTD Ser5-PO₄ ligand. The effects of the alanine substitutions on the stimulation of enzyme-GMP adduct formation by CTD Ser5-PO₄ paralleled the effects on enzyme binding. The T4A peptide was as effective an activator as the wild type peptide, as gauged by the extent of activation (2.5-fold at 2.5 µM T4A peptide) and the concentration dependence of stimulation (half-maximal at 0.3 µM T4A peptide) (Fig. 6). The Y1A mutant peptide, which did not bind to Mce1(211-597), also failed to stimulate the guanylyltransferase activity at concentrations up to 5 µM Y1A (Fig. 6 and data not shown). The P3A and P6A mutants, which displayed intermediate binding in the affinity chromatography assay, also showed diminished affinity for the allosteric effector site on Mce1(211-597), as revealed by the shifts to the right in their respective activation profiles; the guanylyltransferase activity at 2.5 µM P3A or P6A was less than half of that at the equivalent level of the wild-type peptide (Fig. 6). Raising the P3A and P6A concentrations to 5 µM elicited a further increase in the guanylyltransferase activity (not shown), to about the levels achieved at 0.6 and 0.4 µM of the wild-type CTD Ser5-PO₄ peptide. We estimate the P3A and P6A mutations diminish affinity for the allosteric site by factors of about 8 and 10, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Independent Recruitment of S. pombe Triphosphatase and Guanylyltransferase to the Transcription Elongation Complex-- The present study highlights a distinctive strategy of cap targeting in fission yeast whereby the triphosphatase and guanylyltransferase components of the capping apparatus do not interact physically with each other (as they do in budding yeast and metazoans), but instead bind independently to the phosphorylated CTD. The capacity of S. pombe RNA triphosphatase to bind to the CTD in vivo and in vitro contrasts with apparent inability to detect direct interactions of the S. cerevisiae or mammalian triphosphatases with the CTD (8, 12). How then to explain the observed genetic interaction of S. pombe Pct1 with Pce1 for functional complementation of cap formation in S. cerevisiae? Recent studies suggest that the essential interaction in trans between the S. cerevisiae guanylyltransferase Ceg1 and triphosphatase Cet1 confers important functional advantages on Ceg1, by antagonizing negative effects of CTD-PO₄ binding on the guanylyltransferase activity of Ceg1 (8) or by protecting the inherently labile Ceg1 protein against thermal inactivation at physiological temperatures.² Because the S. pombe triphosphatase Pct1 lacks the high-affinity Ceg1-binding domain responsible for these salutary effects on Ceg1 function (11), it is most likely that the loss of Ceg1 function is responsible for the failure of Pct1 by itself to complement the cet1 mutation. However, co-expression of Pct1 with Pce1, which is inherently thermostable² and is not inhibited by CTD-PO₄ (present study), circumvents the need for a Ceg1-binding domain on the triphosphatase and allows functional complementation of the first step of capping by Pct1.

Importance of CTD Length and Phosphorylation Array for CTD Effector Functions-- The observation of a minimal CTD length for interactions with S. pombe Pct1 in vivo, with partial phenotypes at the immediate supra-threshold repeat lengths, and "normal" interactions with about half of the full-sized S. pombe CTD, mirrors to a considerable degree the overall pattern of CTD length effects on yeast cell growth (17, 18). The threshold length for the CTD two-hybrid interactions with S. pombe Pce1 is shifted upward by several heptad repeats compared with the requirements for S. cerevisiae cell growth. To our knowledge, the effects on fine incremental changes in CTD length in S. pombe Rpb1 on the growth of S. pombe have not been reported. Nevertheless, it is reasonable to think that at least some of the limitations on cell growth that accompany CTD truncation pertain to disruption of key contacts with the capping apparatus. Suppression analysis of CTD deletions in S. cerevisiae point clearly to interactions with the Srb complex as a limiting determinant of CTD function during transcription initiation (27). Yet, the finding that sublethal CTD truncations exacerbate the phenotypes of S. cerevisiae ceg1-ts mutants (4) argues that capping enzyme recruitment during elongation can also be limiting for cell growth.

The higher CTD length requirements for capping enzyme interactions in vivo in a two-hybrid assay versus in vitro with synthetic CTD phosphopeptides suggest that CTD length effects on CTD phosphorylation may influence the in vivo readout. It is also possible that the accessibility of the CTD to the capping enzymes in vivo in the two-hybrid assay is affected by the presence of additional proteins and macromolecular assemblies that compete for binding sites on the CTD, with the result being that a longer CTD is needed for the two-hybrid interactions with the capping enzymes.

Truncation of the mammalian pol II CTD to a length of only 5 heptad repeats leads to a significant reduction in the efficiency of mRNA capping in mammalian cells (2). Given the high affinity of the mammalian capping enzyme for CTD Ser5-PO₄ or Ser2-PO₄/Ser5-PO₄ peptides containing 4 heptad repeats, and the fact that Mce1 binds, albeit with lower affinity, to a CTD Ser5-PO₄ peptide containing only two heptads (12), it is again possible that the in vivo length restriction on CTD-directed mammalian capping reflects requirements to attain an adequate CTD phosphorylation array. Indeed, the Cdk7-cyclin H-p36 complex preferentially phosphorylates longer CTD substrates with multiple YSPTSPS repeats (28). However, it is also possible that the accessibility of the truncated CTD in vivo in the context of the mammalian transcription elongation complex is limited by the structure of RNA polymerase II per se, the presence of protein factors associated with the polymerase (although not necessarily with the CTD), and the competition among CTD-binding proteins for the shortened CTD.

Ser-5 and Ser-2 are both extensively phosphorylated in vivo and various CTD serine kinases differ in their site preference (29-31). Here we found that binding of the S. pombe triphosphatase to the CTD is enhanced when both Ser-2 and Ser-5 are phosphorylated compared with Ser-5 phosphorylation alone. A similar enhancement of binding to the doubly phosphorylated CTD versus singly phosphorylated CTD was reported for the cap methyltransferase of C. albicans (22). However, Ser-2 phosphorylation did not significantly affect the binding of S. pombe Pce1 to the Ser5-PO₄ CTD and we saw only a modest increase in binding of the mammalian guanylyltransferase to the doubly phosphorylated CTD, with no increase in the amplitude of the allosteric CTD Ser5-PO₄ effect.

We show here that the S. pombe capping enzymes Pct1 and Pce1 can discriminate between Ser2-PO₄ and Ser5-PO₄ CTD ligands containing the identical number of heptads and phosphate moieties; Pct1 did not bind at all to the CTD phosphorylated on Ser-2 under the conditions tested, whereas Pce1 bound less avidly to the Ser2-PO₄ CTD than to the Ser5-PO₄ form. Functional distinctions between the Ser-2 and Ser-5 positions had been suggested previously by the identification of extragenic suppressors of S2A mutations of the CTD heptad and the finding that they are incapable of suppressing S5A mutations (32) and by the disparate allosteric effects of Ser-2 and Ser-5 phosphorylation on the mammalian guanylyltransferase (12). It is conceivable that the differences between the Ser-2 and Ser-5 phosphorylation arrays with respect to CTD interactions with capping enzymes would be less marked if the number of Ser2-PO₄ heptad repeats was increased significantly. The technical problems of synthesizing and isolating a defined homogeneous CTD phosphopeptide of, for example, 10 or more heptad repeats with an identical phosphorylation site in each heptad hamper an evaluation of this scenario.

Dissecting the Importance of CTD Primary Structure for CTD-PO₄ Effector Functions-- The CTD of elongating pol II serves as a landing pad for multiple macromolecular assemblies that regulate mRNA synthesis and processing (14). The capping apparatus is the first of the assemblies to act on the nascent pre-mRNA and the one for which binding of the catalytic components is most clearly dependent on CTD phosphorylation. Dynamic remodeling of the CTD phosphate array in the elongation complex by kinases and phosphatases can thereby influence the efficiency and timing of capping and the dissociation of the capping enzymes from the elongation complex (7, 33). But how does the CTD structure itself dictate interactions with the RNA modifying enzymes independent of the phosphorylation state? Using mutated versions of the CTD Ser5-PO₄ peptide, we uncovered structure-activity relationships for the non-phosphorylated side chains in CTD-PO₄ interactions with fission yeast and mammalian capping enzymes.

First, we established that the Thr-4 side chain plays no apparent role in Pce1, Pct1, or Mce1 binding and is not critical for Mce1 activation. These results are remarkable given that the threonine side chain is present in 44 of 52 repeats of the mammalian CTD and 26 of 29 repeats of S. pombe CTD, but they are consistent with the recent report that S. cerevisiae is viable when every one of the Thr-4 positions in its CTD was replaced by alanine (34).

Second, we showed that Tyr-1 is essential for the interaction of the phosphorylated CTD with Pce1, Pct1, and Mce1. It had been established previously that Tyr-1 was essential for S. cerevisiae growth (17), but it was not clear if Tyr-1 mutations were lethal because: (i) they prevent tyrosine phosphorylation, (ii) Tyr-1 is important for serine phosphorylation, or (iii) Tyr-1 is critical for one or more essential CTD interactions independent of confounding phosphorylation effects. The experiments herein do not bear on the first two issues, but our data are consistent with the third scenario. Although binding to Pce1, Pct1, and Mce1 was affected by the Y1A change, we cannot infer whether the loss of function was caused by an intramolecular perturbation of CTD-PO₄ structure or the loss of a key intermolecular contact between Tyr-1 and the CTD-PO₄-binding site on the capping enzymes. Tyr-1 is present in all of the 52 heptad repeats of the mammalian CTD and in all 29 repeats of the S. pombe CTD.

Third, we find that alanine substitution for Pro-3 had a more severe impact on interaction with Pce1 and Pct1 (elimination of detectable binding) than it did on Mce1 (reduced affinity in binding and activation). Conversely, the P6A mutation affected binding and activation of Mce1, but had little impact on Pce1 binding, while eliminating binding of Pct1. These results indicate that the structural basis for interaction with the phosphorylated CTD is at least somewhat variable between capping enzymes from different species. We anticipate that synthetic phosphopeptides containing defined modifications will provide powerful tools to dissect the structural requirements for recognition of the phosphorylated CTD by other RNA processing enzymes and macromolecular complexes.

	ACKNOWLEDGEMENTS

We thank San San Yi for expert peptide synthesis and Julie Cooper for the S. pombe AD-cDNA library.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM52470.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Fax: 212-717-3623; E-mail: s-shuman@ski.mskcc.org.

Published, JBC Papers in Press, May 31, 2001, DOI 10.1074/jbc.M102170200

² S. Hausmann, C. K. Ho, and S. Shuman, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: pol II, polymerase II; CTD, carboxyl-terminal domain; BD, binding domain; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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