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J. Biol. Chem., Vol. 282, Issue 3, 2101-2115, January 19, 2007
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1
123456
From the
Department of Molecular Microbiology, Tufts University School of Medicine and the Sackler Graduate School of Biomedical Sciences, Boston, Massachusetts 02111 and the Department of Biochemistry and Department of Chemistry, University of Washington, Seattle, Washington 98195
Received for publication, October 24, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Among the proteins that ensure a communication between transcription and 3'-end processing are human CstF-64 and its Saccharomyces cerevisiae orthologue Rna15, two of the earliest identified components of the polyadenylation machinery (7, 8). These proteins play a key role in mRNA 3'-end processing by recognizing one of the signal sequences that specify poly(A) sites (9, 10). Altering the levels of CstF-64 or mutating Rna15 can shift the pattern of poly(A) site utilization when multiple processing sites are present on the same transcript (11-15). Defects in Rna15 also have consequences on mRNP biogenesis that transcend 3'-end processing, such as hindering transcription elongation and preventing transcription termination downstream of poly(A) sites (16-18). The G/U-rich sequences that represent binding sites for CstF-64 are essential for proper termination of transcription (19), indicating that the functions of Rna15 and CstF-64 in transcription are conserved as well. Mutation of RNA15 also causes poor recruitment of the Yra1 export factor to actively transcribed genes (20), retention of mRNA in the nucleus (18, 21), and poor release of mRNA from the site of transcription (22). The defective mRNPs produced by Rna15 mutant cells are recognized and degraded by the nuclear exosome (23).
The Rna15 and CstF-64 proteins are closely related in sequence and structure at their N and C termini (Fig. 1A). These two proteins are also homologous to Pti1 (24-26), a component of the APT complex involved in S. cerevisiae snoRNA 3'-end formation (26). These three proteins contain RRM-type RNA binding domains at their N termini that are both necessary and sufficient for RNA binding (27-29). Mutation of conserved amino acids in this motif within Rna15 impairs the ability of Rna15 to interact with RNA and to function in mRNA 3'-end processing (10). Metazoan CstF-64 proteins have a conserved 100 amino acid region called the "hinge" immediately C-terminal to the RRM that is also found in Pti1 and Rna15 (24). This region of CstF-64 interacts with CstF-77 and with Symplekin, a protein implicated in the assembly of several RNA 3'-end processing machineries (30-34). The hinge domain of Pti1 interacts with Rna14 and Pta1, the yeast orthologues of CstF-77 and Symplekin, respectively (24). The central region of CstF-64 is variable in length and composition, and its function is unknown. In most metazoans, it is rich in proline and glycine and contains up to a dozen repeats of the MEAR(A/G) motif, which are missing altogether in worms and fungi (32).
The 50 amino acids at the very C terminus of the Rna15/CstF-64 family are even more highly conserved than the N-terminal RRM. A mutant of the Schizosaccharomyces pombe homologue, Ctf1, lacking the last 70 amino acids (including this C-terminal domain) caused transcription to continue downstream of the poly(A) site, leading to the proposal that this region participates in termination of transcription (35). Consistent with this idea, the last 100 or so amino acids of Rna15/hCstF-64, and of Ctf1, were shown genetically and biochemically to interact directly with the transcription factors Sub1/PC4 and Res2 (35, 36). This region in Ctf1 is thought to function only in transcription termination and not in 3'-end processing, because its loss does not affect accumulation of properly processed mRNA in vivo (35). However, the precise role of the Rna15/CstF64 C terminus in cleavage and polyadenylation has not been addressed.
Rna15 is part of CF IA (37, 38) and interacts directly with the Rna14 and Pcf11 subunits of this factor (39), and with the poly(A)-binding protein Pab1 (40). The interaction between Rna15 and Pcf11 is particularly interesting in light of recent reports that provide a role for the N terminus of Pcf11 in the promotion of transcription termination (41, 42). However, the domains of Rna15 important for the contacts with Rna14 and Pcf11, and the role of these interactions in 3'-end processing and termination, have not been delineated. The three-dimensional organization of the regions of the protein beyond the RRM also remains to be determined.
In this study, we have investigated the interaction of the C-terminal half of Rna15 with Rna14, Sub1, and Pcf11. We report that the C-terminal domain of the Rna15/CstF-64 protein family forms an unusual structure of three helices that positions highly conserved residues on the protein surface to mediate protein/protein interactions. We demonstrate that this highly conserved domain functions in the cleavage and polyadenylation of mRNA precursors by providing a contact site for Pcf11, whereas the region of the protein just N-terminal to it interacts with Rna14 and Sub1. These studies provide structural and biochemical insight into highly conserved protein/protein interactions responsible for assembly of the 3'-end processing apparatus in all eukaryotes.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The RNA15-His6 coding sequence from plasmid pET21b/Rna15 (37) was subcloned into the LEU2 vector Yeplac181 to generate Yeplac-RNA15H6. Truncations of the RNA15 open reading frame from the 3'-end were amplified using the primers listed in Table 1 and cloned into pET21b (Novagen) between the NdeI and XhoI sites. The mutants rna15-CTD1 and rna15CTD2 were constructed using a forward PCR primer (supplemental Table 1) containing the desired mutation and a reverse primer corresponding to the end of the RNA15 coding sequence. The wild-type BamHI/NdeI fragment on pET21b/Rna15 was then replaced with the mutated PCR fragment digested with BamHI and NdeI. Mutations were confirmed by sequencing. BclI-BlpI fragments from these mutant rna15 constructs were subcloned into BclI-BlpI-treated Yeplac-RNA15H6. This was accomplished by first growing the plasmids in the dam-Escherichia coli strain UF253. All of the Yeplac-RNA15H6 variants were transformed into LM31 for expression in yeast and phenotypic studies. Additional truncations of Rna15 (t1, t2, t3, t4, and t5, see supplemental Table I) were also made. These behaved identically to rna15-t6 in terms of cell viability and interactions with RNA, Pcf11, Rna14, and Sub1 (data not shown). The plasmids pET21b/RNA14 (37) and pRSETB-SUB1 (36) were used for in vitro translation of Rna14 and Sub1. The Rna15/His6-Rna14 complex was expressed using the pET-Duet-1 plasmid (Novagen), a gift from I. Taylor (45). To express Pcf11, the PCF11 coding sequence was cloned into the pMAL vector (New England Biolabs) to give pMAL-PCF11. For purification of Sub1 from yeast, the SUB1 open reading frame was cloned into the pYES2CT vector (Invitrogen), which introduced the V5/His6 tag onto the C terminus and allowed galactose-induced expression from a high copy plasmid.
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-mercaptoethanol, 10% glycerol, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM pepstatin, and 0.6 mM leupeptin. The cell lysate was allowed to bind to 1 ml of Talon resin (Clontech) or nickel-nitrilotriacetic acidagarose (Qiagen) for 1 h at 4°C with gentle agitation. Washings were performed for 10 min each for two times in wash buffer 1 (binding buffer plus 5 mM imidazole (pH 7)), 10 min each for two times in wash buffer 2 (wash buffer 1 lacking 0.1% Nonidet P-40), and 10 min each for two times in wash buffer 3 (same as wash buffer 2 but with 125 mM KCl). Proteins were eluted with 100 mM imidazole in wash buffer 3. The eluate was dialyzed against buffer containing 20 mM Tris (pH 8.0), 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol, 1 mM PMSF, 2 mM pepstatin A, and 0.6 mM leupeptin with two buffer changes for 1 h each (3 h total). Pcf11 was expressed as an MBP fusion from pMAL-PCF11 in E. coli and purified on amylose resin following the protocol provided by New England Biolabs. The His6-Rna14/Rna15 heterodimer was expressed as described by Noble et al. (45) and purified by nickel affinity chromatography. Sub1-V5/His6 was purified from the yeast strain INVSc1 (Invitrogen) transformed with pYES-Sub1, in which expression was induced by growth in selective medium containing 2% galactose. Cells were lysed with the yeastBuster protein extraction reagent (Novagen) according to the manufacturer's protocol, and Sub1-V5/His6 was purified by nickel-affinity purification (Qiagen) using a wash buffer containing 500 mM NaCl. 35S-Labeled Rna14 and Sub1 proteins were made by in vitro coupled transcription and translation (TNT) reactions using the rabbit reticulocyte lysate system (Promega) in the presence of [35S]methionine (Amersham Biosciences) as described by the manufacturer. One microgram of plasmid was used in each TNT reaction.
For structural investigations, different CstF-64, Rna15, and PC4 protein constructs were subcloned into pET-28 (Novagen). Proteins were overexpressed in BL21(DE3) cells or Rosetta (DE3) pLysS strains (Novagen) by induction with 1 mM isopropylthiogalactoside at a cell density of 0.5 OD. Expression was conducted in M9 minimal media containing D-[13C]glucose and/or [15N]ammonium chloride, as needed. The CstF-64 and Rna15 constructs were purified by Ni2+ affinity chromatography. Following digestion with thrombin to remove the hexahistidine tag, the solution was filtered and reloaded onto the Ni2+ affinity column. The protein-containing flow-through was concentrated and further purified by gel filtration. No detectable impurities were found for any of the constructs investigated by gel electrophoresis or matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. Because of relatively poor protein expression for Rna15, the structural analysis was focused on CstF-64.
Full-length and N-terminal truncated (residues 1-63) PC4 proteins (supplemental Table II) were phosphorylated in vitro with casein kinase II (New England Biolabs). Each 10-µl phosphorylation reaction contained 0.2 mM protein, 20 mM Tris-HCl (pH 7.5), 150 mM KCl, 10 mM MgCl2, 1 mM ATP, and 10 units of casein kinase II. The reactions were incubated at 30 °C for 30 min and quenched with 10 µl of SDS loading solution. Phosphorylation was confirmed by SDS-PAGE analysis and by mass spectrometry, which showed a shift of 380 Da above the unphosphorylated PC4 peak, indicating that five phosphates on average were incorporated into PC4.
Pulldown Assays—To test the interaction with Rna14 and Sub1, 5 µg of Rna15 protein (or its truncated derivatives) were bound to 15 µl of Talon beads (Clontech) for 1 h at 4 °C in 200 ml of buffer containing 20 mM Tris (pH 8.0), 150 mM KCl, 5% glycerol, and 0.01% Nonidet P-40 (IP-150). After 1 h, 15 µl of His6 peptide (10 mg/ml) was added, and the proteins were incubated for 1 h at 4°C. 10 µl of the TNT reaction containing 35S-labeled Rna14 was then added, along with an additional 15 µl of H is 6 peptide, 2 units of RNase A, and 10% (v/v) fetal calf serum to reduce nonspecific binding. Following incubation on a rotating shaker at 4 °C for 1 h, the beads were washed four times in IP-150. The bound products were analyzed by SDS-PAGE. Radioactive proteins were visualized on a Storm 860 PhosphorImager (GE Healthcare), whereas Rna15 protein variants were identified by silver staining. A similar procedure was followed for pulldowns with Sub1, but 1 µg of Rna15 protein or its truncations were used, and the beads were saturated by 20 µl of H is 6, followed by addition of 10 µl of the TNT reaction containing 35S-labeled Sub1. The binding of the second protein to Talon beads in the presence of saturating amounts of His6 peptide was used as a background control.
To assay the interaction of Rna15 and Pcf11, 20 µl of a 50% slurry of amylose resin (New England Biolabs) was pre-washed and incubated with MBP-Pcf11 (4 µg) for 1 h in 200 µl of IP-150 buffer at 4 °C. The beads were washed three times with 1.4 ml of IP-150 and then resuspended in 200 µl of IP-150 containing 10% fetal calf serum. Wild-type or mutated Rna15 (4 µg) proteins were then added and incubated on a rotating shaker for 1 h at 4 °C, followed by four washes with IP-150 buffer. The bound products were resolved by SDS-PAGE and visualized by immunoblotting with Rna15 polyclonal antibody; MBP protein (instead of MBP-Pcf11) was added for all control reactions.
Pulldowns with Sub1-V5/His6 were performed by binding Sub-V5/His6 to anti-V5 antibody (Invitrogen) on recombinant protein G-agarose beads (Invitrogen), and proteins detected by immunoblotting with the Rna15 polyclonal antibody.
Poly(U) Binding Assays—Poly(U)-Sepharose beads (Amersham Biosciences) were washed and equilibrated according to the manufacturer's instructions prior to use in order to remove any unbound poly(U). 20 µl of beads were then bound to 1 µg of recombinant Rna15 (or its truncations) at 4 °C for 1 h in 1ml of PU buffer (20 mM Tris-Cl (pH 8.0), 0.1% Nonidet P-40, and 100 mM KCl). Bovine serum albumin was used as a negative control. Unbound proteins were removed by three 1-ml washes of PU buffer. Bound proteins were resolved by SDS-PAGE and visualized by silver staining.
RNA Processing Assays and ChIP Analysis—Yeast cell extracts were prepared as described previously (46). Capped, full-length, or pre-cleaved 32P-labeled RNAs were prepared by runoff transcription from plasmid pJCGAL7-1 digested with AvaI or plasmid pJCGAL7-9 digested with NsiI, respectively, as described previously (46, 47). Each reaction was performed in a volume of 10 µl containing 1 mM ATP, 10 mM creatine phosphate, 1 mM magnesium acetate, 75 mM potassium acetate, 2% polyethylene glycol 8000 (PEG 8000), 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin (New England Biolabs), 0.4 units of RNasin (Promega), 10 nM radioactive RNA precursor (
250,000-280,000 cpm), and 30 µg of yeast cell extract. Reactions were prepared on ice and incubated first at 4 °C for 10 min and then at 30 °C for 20 min. Reactions were stopped by the addition of proteinase K and SDS, brought to a volume of 30 µl with Tris-EDTA (pH 7.5), and extracted once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v). One-tenth of the reaction was resolved on a 5% acrylamide, 8.3 M urea gel and then visualized by using a PhosphorImager. For ChIP analysis, in vivo cross-linking with formaldehyde, preparations of chromatin and immunoprecipitations were performed as described previously (4) using 7 µl of 
-Rpb3 antibodies (Neoclone Biotechnology) preincubated with protein A/G PLUS-agarose beads (sc-2003; Santa Cruz Biotechnology).
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and 
were obtained from the statistical analysis of 13C chemical shifts as implemented in TALOS (53). Structure calculations were executed by torsion dynamics in CYANA (version 1.06). All structures having no distance restraint violation larger than 0.2 Å or no violation of dihedral angle constraints larger than 5° were accepted (90% of all calculated structures). Rna15 and Pti1 C-terminal domains were modeled from the atomic structure of the homologous CstF64 domain using the in silico mutagenesis tools of the program MOLMOL (54). Interaction between PC4 and CstF-64—NMR was also used to study the interaction of the C-terminal domain of human CstF-64 with various forms of unphosphorylated and phosphorylated human PC4. HSQC spectra were typically recorded at 300 K, but in several cases data were duplicated at 290 K as well. Titration experiments were conducted using the sample combinations shown in supplemental Table II at about 0.3 mM 15N-labeled protein by recording an HSQC of the labeled free protein. A second HSQC was then recorded under identical conditions after adding the unlabeled binding partner in slight excess. Buffers were 50 mM sodium acetate, 300 mM NaCl, 4 mM DTT (pH 5), except in one case when the following buffer was used to exactly duplicate the conditions reported by Calvo and Manley (36): 20 mM HEPES-Na (pH 7.9), 100 mM NaCl, 10% glycerol, 0.5 mM DTT, 0.2 mM EDTA, 0.2 mM PMSF.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The C-terminal Domain of Rna15/CstF-64 Forms a Novel All-helical Structure—In pursuing structural investigations of the Rna15/CstF-64 C-terminal domain, we focused on the human protein because expression levels were considerably higher compared with Rna15. Poor expression of this region of Rna15 has made it very difficult to obtain sufficient amounts to determine its structure by NMR. Although the sequence analysis shows that only the last 40 or 50 residues of CstF-64/Rna15 are conserved (Fig. 1B), we started our structural investigation with a slightly larger region (504-577 of the human sequence) based on biochemical studies of the interaction with PC4. When a similar fragment was removed from CstF-64, the interaction with PC4 was abolished (36). The 15N-HSQC spectrum of this construct shows that approximately half of the signals have sharp line widths, negative 1H-15N heteronuclear NOE (data not shown), and very poor dispersion (Fig. 2A). The remaining signals show instead broader line widths, positive NOEs, and much better dispersion. These observations suggest the presence of a structured region next to an unstructured N-terminal fragment. Because the characteristic Gly signals largely belong to the unstructured part of the domain, it is clear that the 504-529 fragment of the construct, containing nearly all of the Gly residues, is the unfolded region. These domain boundaries agree well with the pattern of conservation, which decays sharply before position 530, and with the secondary structure prediction (Fig. 1B). Signals in the 15N-HSQC spectrum of a shorter construct (531-577) overlap with the well dispersed signals in the longer construct (Fig. 2A), confirming that the last 46 residues form a structurally independent domain and that truncation of the segment 504-529 does not affect the structure of the C-terminal domain. These results prompted us to proceed with the structural studies on the construct, including only residues 531-577. The lack of aromatic residues (Tyr, Phe, or Trp) compromises the dispersion of the NMR spectra even for this shorter construct. Thus, 13C- and 15N-edited NOESY spectra had to be used in order to assign NOE data unambiguously and generate a substantial number of medium and long range NOEs constraints (Table 1).
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-helix orthogonal bundle (Fig. 2, B-D) with two longer helices (almost four turns in length for helices 1 and 3) connected by a shorter helix 2 (two turns). The fold of the domain is maintained by a well defined network of interactions. Helices 1 and 3 contact each other through a hydrophobic interaction between residues Leu564 (in helix 3) and Lys537 (in helix 1), reinforced by contacts between His535 and Gln561 on the opposite side of helix 1 (Fig. 2D). Although residues involved in hydrophobic interaction are totally conserved throughout all CstF-64 homologues, the hydrophilic contacts are more variable. Together, these interactions maintain the angle between the axes of the two helices at the unusual value of 50° ± 5° (Fig. 2C). Helix 2 lies on top of the forked structure defined by helices 1 and 3, and it forms a small hydrophobic cluster with them that further stabilizes the structure of the domain. Because of the small size of the protein, part of the hydrophobic core is solvent-exposed, and only a handful of residues display solvent accessibilities below 10% (mean values over the 20 structure ensemble): Ala538 (1.6%), Ile541 (10.8%), Met542 (7.1%), Ile552 (2.9%), Ser562 (7.9%), and Ile563 (5.1%). Ile552 is one of the least accessible and also one of the seven residues absolutely conserved among different homologues. Positions 541 and 563 conserve their hydrophobic character, whereas the long yet linear side chain of Met542 is replaced by Lys in Rna15 and Pti1. The introduction of a positive charge might be thought of as a destabilizing mutation, but a more detailed examination of the environment of Met542 reveals that its methyl group is solvent-exposed and therefore can be substituted with Lys without destabilizing the protein fold. Analysis of the sequence homology data in light of this structure strongly suggests that all CstF-64 C-terminal domain homologues will have the same structure. For example, the key contacts between helices 1 and 3 (Lys537-Leu564 in CstF-64) are maintained in Rna15 (Lys-Trp) and Ctf1 (Arg-Leu), but they are changed to Gln-Glu in Pti1. Although these two side chains retain the ability to interact across the helix 1-helix 3 interface (i.e. by hydrogen bond), this and other differences with CstF64, Rna15, and Ctf1 suggest that Pti1 is a more distant homologue of these proteins.
The structure of the CstF-64 C-terminal domain allowed us to precisely define domain boundaries that agree well with sequence and prediction data (Fig. 1B). By using this information to design a new expression construct, we obtained sufficient amounts of the unlabeled yeast protein to determine a NOESY spectrum of the C-terminal domain of Rna15. This spectrum contains the characteristic signature expected for a helical protein (supplemental Fig. 1) as follows: amide to aliphatic NOEs consistent with a folded domain and the characteristic N-N NOEs typical of a helical protein. The low concentration of the sample because of poor protein expression resulted in a suboptimal spectrum. Nevertheless, these results, coupled with the sequence homology, unambiguously support the structural homology between the C-terminal domains of CstF-64 and Rna15.
Remarkably, out of seven absolutely conserved residues within the C-terminal domain of CstF-64, only one has an indisputable structural role (Ile552), implying that the remaining six will have important functional roles. A revealing picture emerges when these conserved residues are mapped onto the three-dimensional structure of the domain (Fig. 3A). Five conserved hydrophobic residues (Val544, Leu545, Leu547, Leu555, and Pro556) form a continuous solvent-exposed patch flanked by two Gln residues (543 and 551), which are also conserved. A comparison of solvent accessibilities across conserved hydrophobic residues provides some insight into the possible role of these residues. As mentioned before, Ile552 is deeply buried in the structure (solvent accessibility 2.9%), suggesting an architectural role for this residue. The other conserved hydrophobic residues are much more exposed; for example, Leu547 has on average 62% of its side chain exposed. The conservation of this hydrophobic patch strongly suggests that it might form a protein interaction interface. The conservation of the two Gln residues is also unrelated to any structural stabilization role, suggesting that they function in providing interaction specificity as well. Finally, it is also interesting that the only absolutely conserved positive residue, Lys567 (see above), is placed near this putative interaction surface.
The C Terminus of Rna15 Is Critical for Cell Growth—The evolutionary conservation of the key residues discussed above maintains an architecture that must be important for the functional role of this domain. To probe the function of this conserved C-terminal domain, Rna15 was tagged with His6, and sequential deletions of 17-19 amino acids were made from the C terminus (Fig. 1A). These truncations were expressed in a strain carrying a wild-type RNA15 gene on a URA3-containing plasmid and a deletion of the chromosomal copy of RNA15. These strains all grew as well as wild-type cells, indicating that none of the shorter forms of Rna15 exerted a dominant negative effect on cell fitness. All of the mutant forms were stably expressed at levels comparable with the wild-type protein (Fig. 4A).
The truncated Rna15 proteins were then tested for their ability to provide Rna15 function in vivo, using plasmid shuffling to remove the covering plasmid containing the RNA15 gene. The first two deletions (rna15-t10 and rna15-t9), which remove helix 3 or helices 2 and 3 and destroy the C-terminal domain structure, could not support cell growth. Surprisingly, removal of the entire C-terminal domain (rna15-t8) resulted in a viable rna15 mutant, albeit one that grew slowly at 24 °C and exhibited thermosensitive lethality at 37 °C (Fig. 4B). The larger deletions found in rna15-t7 and rna15-t6 resulted in mutants that could not grow at either temperature.
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Defects in the mRNA 3'-end processing machinery can cause poor transcription termination downstream of a poly(A) site (1, 2). For example, the rna15-1 mutant that is defective for in vitro cleavage and polyadenylation (7) also shows defective termination in transcription read-through assays (17). Chromatin immunoprecipitation (ChIP) has been used recently as an assay for termination defects, using as a read-out the persistence of RNAP II in regions of a gene located beyond the poly(A) site (4). We used ChIP to determine whether the rna15-t8 allele was impaired for transcription termination. In this experiment, the amount of RNAP II found on different portions of the highly transcribed ADH1 gene was assessed by immunoprecipitation of cross-linked chromatin with antibody against the RNAP II Rpb3 subunit, followed by PCR amplification to detect the pulldown of fragments representing different regions of the ADH1 gene (Fig. 4D). The RNAP II signal from wild-type cells grown at 37 °C is strong just beyond the poly(A) site (PCR product 2) but drops markedly at a position 300 bases further downstream (PCR product 3). A decline similar to what is seen with wild-type cells is observed for the rna15-t8 mutant, indicating that termination in this mutant at the nonpermissive temperature is as efficient as wild-type cells. In contrast, the termination-defective pcf11-9 mutant (44) shows an increase in signal from the PCR product 3, in agreement with other ChIP analyses using this mutant (5).8 From these experiments we conclude that rna15-t8 is defective for mRNA 3'-end processing but normal for poly(A)-site dependent termination.
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The same Rna15 proteins were then used in pulldown assays using nickel-affinity beads and in vitro translated Sub1 and Rna14 (Fig. 5, C and D). In agreement with previous studies (36, 39), Sub1 and Rna14 interact with wild-type Rna15. The shortest form of Rna15 that binds to Sub1 and Rna14 is rna15-t7; binding drops to background levels with rna15-t6. This experiment indicates that the interactions with Sub1 and Rna14 require a region between amino acids 208 and 225, in agreement with Calvo and Manley (36), who mapped the Sub1 interaction to amino acids 191-290 of Rna15.
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The C-terminal Domain of CstF-64 Is Not Involved in Interaction with the Sub1 Homologue, PC4—A two-hybrid interaction between CstF-64 and PC4 was previously shown to require the last 63 amino acids of CstF-64 (36). Our analysis of the comparable region of Rna15 described above shows that the highly conserved domain at the C terminus is not necessary for this interaction and suggests that the region of CstF64 critical for PC4 interaction lies instead outside of the C-terminal domain. To further probe for an interaction between the very C terminus of CstF-64 and PC4, we used NMR, which is capable of detecting even weak interactions (i.e. dissociation constants in the millimolar range) that could become significant when proteins are assembled into physiologically relevant complexes. We probed PC4 with two different C-terminal fragments of human CstF-64, one containing the last 63 amino acids and a second containing the most highly conserved 40 amino acids at the very C terminus (the fragment used for structure determination).
The analysis of the 15N-HSQC NMR spectrum of full-length PC4 was consistent with a structured domain at the C terminus (57, 58), whereas the resonances corresponding to the N-terminal fragment indicated an unfolded polypeptide. We then mixed 15N-labeled PC4 with unlabeled CstF-64, and vice versa, in several possible combinations (supplemental Table II), but the data revealed no significant spectral changes (data not shown). Reasoning that the DNA-binding domain may perhaps inhibit CstF-64 binding, we mixed the N-terminal fragment of PC4 with human CstF-64, without observing any changes at all. Finally, we used casein kinase II to phosphorylate PC4 in vitro, but we again failed to detect any interaction with CstF-64. The absence of chemical shift changes indicates very strongly that these two protein domains do not physically interact and that the CstF-64/PC4 interaction previously observed in vivo or in cell extracts (36) is mediated by a different domain of CstF-64 or indirectly by other proteins.
Pcfll Interacts with the Conserved C Terminus of Rna15—In addition to Rna14 and Sub1, the Pcfll subunit of CF IA also makes direct contact with Rna15 (39). To map the region of Rna15 required for the interaction with Pcf11, we used recombinant MBP-tagged Pcf11 bound to amylose beads as bait for the Rna15 variants (Fig. 6A). The interaction with Pcf11 was lost with the minimal truncation, rna15-t10. This truncation removes the last 16 amino acids spanning helix 3 and destroys the C-terminal domain structure. Therefore, to directly investigate the putative interaction surface identified in the structural analysis (Fig. 3A), we changed the two strictly conserved glutamines in Rna15 at positions 261 and 269 to alanine to give rna15-CTD1. During the construction of this mutant, we also isolated a second mutant (rna15-CTD2) in which three amino acids between these glutamines (in the loop region between helix 1 and helix 2) had been replaced with a single histidine (Fig. 6B). Because these residues are surface-exposed, neither of these mutations is expected to affect the global fold of the C-terminal domain. Both mutants were defective for binding to MBP-Pcf11 (Fig. 6B). The more severe mutation, rna15-CTD2, caused slow growth at 25 and 37 °C compared with wild-type cells, and thermosensitive growth at 38 °C (Fig. 6C). The mutant with only the E261A/E269A replacement showed wildtype growth at 25 °C but was temperature-sensitive when grown in the presence of 1.5% formamide, a reagent known to enhance defects in protein/protein interactions in yeast mutants (59). These results confirm the importance of the conserved residues in this interface for Pcf11 interaction and Rna15 function.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Structure and Function of the C-terminal Domain of Rna15/CstF-64—As shown by the sequence alignment in Fig. 1A, the last 50 or so amino acids represent the most highly conserved stretch in this family of proteins (36). This C-terminal domain in CstF-64 (Fig. 2) forms a three-helix bundle, with an uncommon arrangement of the helices. The conservation of key hydrophobic residues indicates that the same structure will almost certainly form in all its homologues, and this prediction is confirmed by our structural analysis of Rna15 (supplemental Fig. 1) as well as our functional studies of Rna15 truncations. Removal of helix 3 (rna15-t10) or helices 2 and 3 (rna15-t9) destroys the domain structure and causes lethality by inactivating the protein, although complete removal of the domain (rna15-t8) gives a thermosensitive growth phenotype and defective RNA processing in vitro. The inviability of the partial truncations might be due to the remnants of the C-terminal domain interfering with other protein/protein interactions.
Destruction of the C-terminal domain of Rna15 correlates with loss of Pcf11 interaction. Pcf11 is important in the structural organization of the mRNA 3'-end processing complex because of its contacts with RNAP II (63), with the Rna14, Rna15, and Clp1 subunits of CF IA (39), and with several components of CPF (64-68), a multisubunit factor that works in conjunction with CF IA in the maturation of 3'-ends (62). Mutations in Pcf11 are known to inhibit 3'-end processing (44, 68). Our structural analysis of the CstF-64 C-terminal domain revealed a cluster of surface-exposed residues (Fig. 3A) that we propose to be an evolutionarily conserved protein/protein interaction surface. The defects of the rna15-CTD1 and rna15-CTD2 mutants, which specifically target this hypothetical interface, confirm the importance of this region of the protein for Pcf11 recognition and Rna15 function.
Pcf11 interacts directly with the RNAP II CTD through a conserved region of Pcf11 that maps to amino acids 1-266 (69, 70). A mutation in Pcf11 that disrupts this interaction prevents transcription termination but not 3'-end processing (44), and a fragment of Pcf11 containing its CTD-binding domain has been shown recently to dismantle the RNAP II elongating complex (41, 42). A different region of Pcf11 (residues 288-400) is required for its interaction with Rna15 (44, 68). The studies cited above have led to the conclusion that the function of Pcf11 in transcription termination is independent of its function in cleavage and polyadenylation. Our finding that loss of an Rna15/Pcf11 interaction severely cripples 3'-end processing but has no effect on termination adds additional support to this model. Cleavage-defective, termination-normal phenotypes have been found with defects in other mRNA 3'-end processing factors, such as when Ssu72 is depleted (66) or for the brr5-1, pta1-2, pcf11-2, or ssu72-2 alleles (44, 71, 72). Thus, although cleavage at the poly(A) site by the 3'-end processing machinery is thought to facilitate RNAP II termination by providing an entry site for the Rat1 5'-3'-exonuclease (73), this may be only one of several convergent events leading to the release of RNAP II from the DNA template (5, 74). Our study suggests that a 3'-end complex lacking the Rna15 C-terminal domain makes contacts with the elongating RNAP II that are sufficient to signal transcription termination, even if the nascent transcript is not cleaved.
The CstF-64 C-terminal Domain Folds in Other Proteins—The unusual 50° angle between helices 1 and 3 of the CstF-64 C-terminal domain observed here has not been reported previously. However, application of the DALI algorithm (75) shows that this domain has some structural similarity with parts of two other proteins as follows: the mammalian Dia1 Formin protein involved in cytoskeletal remodeling (Protein Data Bank code 1Z2C-B; Z score = 3.1) (76) and the RNA silencing suppressor p21 protein from Beet Yellows virus (Protein Data Bank code 2CWO-A; Z score = 2.5) (77). These structurally related regions form well defined domains within these two proteins (supplemental Fig. 2) with the same helical topology and very similar interhelical angles to those observed in the C-terminal domain of CstF-64. However, the similarity is only at the structural level; only one residue (Leu547) is conserved in the sequence of these three domains. To our knowledge, there is no connection of these proteins to mRNA 3'-end processing that would suggest direct structure-function parallelism.
Interestingly, the structurally related domain of Dia1 mediates interaction with the RhoC GTPase (supplemental Fig. 2 and see Ref. 76). In p21, the homologous domain participates more marginally in a homodimer interface. Rather than being isolated, this subdomain makes contacts with other regions in the p21 monomer (77). The question of whether providing a protein interaction interface is a general characteristic of this type of structure deserves further study, but there is currently not enough structural data to make such a conclusion.
The Function of Regions of Rna15 Adjacent to the C-terminal Domain—We have shown that yeast lacking the C-terminal domain of Rna15 grow more slowly than wild-type cells and die at elevated temperatures, in agreement with the severe processing defect and corresponding to a loss of Pcf11 interaction. Our analysis has also identified two functionally important regions that are immediately adjacent to the C-terminal domain. Removal of 18 amino acids in addition to the C-terminal domain cannot be tolerated, but we do not know the function of this region. The mutant protein with this larger deletion is stable, and the interaction with a second CF IA subunit, Rna14, is not perturbed. The only components of the mRNA 3'-end processing complex known to contact Rna15 are Pcf11 and Rna14, but all possible pairings with Rna15 have not been tested, and a critical interaction with this region may yet be found. Alternatively, it may simply provide a linker between the C terminus and the region responsible for interactions with Sub1 and Rna14.
A further truncation of 17 amino acids reveals a region of Rna15 important for the interaction with Rna14 (Fig. 6D). This interaction is evolutionarily conserved, because CstF-64 of humans, fruit flies, and plants all have been shown to interact with CstF-77, the Rna14 homologue (32, 34, 78, 79). Interestingly, a 44-amino acid stretch of Rna15 (residues 165-208) can be aligned with part of the CstF-64 segment that binds to CstF-77 and known as the hinge domain (24, 34). These amino acids are just upstream of the region defined by our deletion analysis as required for recognition of Rna14, suggesting that the N-terminal boundary of the Rna14 interaction domain extends beyond amino acid 208 of Rna15.
The incorporation of Rna15 and CstF-64 into their respective complexes, CF IA and CstF, positively affects the way these proteins interact with the polyadenylation signal. For example, even though the RRM of CstF-64 on its own is somewhat specific for G/U-rich sequences, the CstF complex has greater affinity and specificity for these regulatory elements (9, 29, 80). A possible explanation for these altered properties of CstF-64 has come from the structure of its RRM. The RNA interface is occluded by a C-terminal helix in the free protein that is removed when RNA binds to the protein (28). Destabilization of this helix to uncover the RRM surface of CstF-64 may be facilitated by the interaction of CstF-77 with the nearby hinge domain. We have found that all of the C-terminal truncations of Rna15 bind RNA with similar efficiency, suggesting that Rna14 or Pcf11 binding is not needed to prevent masking of the RRM by other parts of Rna15. However, we have shown previously that Rna14 modulates Rna15 activity in a different way by forming a bridge between the Rna15 and another RNA-binding protein Hrp1, thus stabilizing the contact of Rna15 with the A-rich yeast signal (10). Formation of a complex containing only Rna14 and Rna15 increases the affinity of Rna15 for RNA (45), indicating that the interaction of just these two proteins can also have a positive effect.
Insight into the function of the Rna14/Sub1 interaction region on Rna15 is provided by the analysis of the previously characterized rna15-2 and rna15-1 alleles. The importance of Rna15 for mRNA 3'-end processing was first established with these mutants (7). The rna15-1 mutant also exhibits readthrough transcription beyond the poly(A) site (17). By sequencing, we have found that both mutants contain a single amino acid replacement of L214P (Fig. 6D), which lies in the middle of a region that we have found to be critical for interactions with Sub1 and Rna14 (residues 208-225). Thus, the processing defect caused by the L214P mutation may result from a destabilization of the Rna14 interaction. Interestingly, with the program BEHAIRPRED (81), this segment is predicted to form a 2:2 -hairpin with a type I' -turn, in which Leu214 would form part of the turn sequence that is crucial for the formation and stability of -hairpin structures (82).
In an earlier study (36), an Rna15 fragment containing only amino acids 191-290 was shown to be sufficient for Sub1 binding, and our studies refine the boundaries of this interaction (Fig. 6D). Our NMR analysis indicates that PC4/Sub1 does not interact directly with the C-terminal domain of CstF-64 even at millimolar concentrations, and our biochemical studies position the Sub1-interacting region of Rna15 N-terminal to the conserved domain. Sub1 can affect transcription initiation through its interaction with TFIIB (83-85) and 3'-end processing though its interaction with the Pta1 subunit of CPF (66). Calvo and Manley (36) proposed that the termination defect of the rna15-1 mutant is a consequence of an enhanced affinity of rna15-1 for Sub1, and that a third function of Sub1 is to prevent premature termination. We have found that Sub1 interacts equally well with Rna15 in complex with Rna14 as it does with Rna15 alone, suggesting that Sub1 does not exert its anti-termination activity by interfering with the interaction of Rna15 with Rna14. We could not detect an endogenous Rna15/Sub1 interaction in pulldown assays using wild-type yeast extract (data not shown), and such an interaction has not been reported in large scale studies using mass spectrometry to analyze yeast protein complexes (86, 87), consistent with a Sub1/Rna15 interaction occurring transiently during transcription elongation. Taking all of these findings into consideration, we propose that the function of Rna15 in processing is mediated by its interactions with both Pcf11 and Rna14, but its activity in transcription termination requires only the interaction with Rna14 and can be modulated by Sub1 binding in close proximity.
Insights into the Two CstF-64 Homologues in S. cerevisiae—Pti1 is an S. cerevisiae protein that is related to Rna15 and co-purifies with CPF (26, 66, 71, 86, 88). Defects in Pti1 affect the 3'-cleavage site choice and abundance of some mRNAs (24), but the primary function of Pti1 may be to adapt the complex to work on other RNAP II transcripts (25). It has been suggested that this protein acts to uncouple cleavage and polyadenylation (25) in coordination with Nrd1-dependent 3'-end formation of nonpolyadenylated snoRNA and small nuclear RNA transcripts (56). Whereas Rna15 has also been implicated in snoRNA/small nuclear RNA termination (56), our analysis of Rna15 supports the idea that Pti1 and Rna15 use different molecular contacts to execute their respective functions in 3'-end formation. A fragment containing the hinge region of Pti1 interacts directly with Rna14 and Pta1 (24). The interaction of Pcf11 with Pti1 is also mediated by this middle region of the protein but not by its C-terminal domain as in Rna15. Moreover, Rna15 does not bind to Pta1 (72).
The difference in Pcf11 interaction might be explained by structural differences between the C-terminal domains of Rna15 and Pti1. The C-terminal domains of all metazoan CstF-64 proteins as well as that of Rna15 contain four conserved positively charged side chains (Lys537, Lys567, Lys572, and Arg560), of which only the position equivalent to Lys567 is conserved in Pti1p (Fig. 1B). Lys572 and Arg560 are placed on the opposite side to the putative Pcf11-binding interface (Fig. 3C) and hence should not have a leading role in recognition. A more likely explanation for the functional differences relies on the topological arrangement of the helices. In CstF-64, the angle between helix 1 and 3 is determined by the contact between the hydrophobic side chains of Lys537 and Leu564. Equivalent residues can be found in other CstF-64 homologues, such as Rna15 and Cft1, but not in Pti1 (Fig. 1B). Changing the nature of this interhelical contact could have significant structural consequences for a small protein domain like this and potentially re-shape the Pcf11 binding interface, making it unable to recognize the Pcf11 protein. It should be also noted that the crucial Lys537 is near the conserved hydrophobic patch and to Lys567; thus it may also have a role in Pcf11 binding through charge interactions mediated by its side chain amine. This comparison indicates that different protein/protein interactions formed by closely related but slightly divergent factors may provide the selectivity needed to direct mRNA and snoRNA precursors to similar but distinct 3'-end formation pathways.
In summary, we have characterized the structure and function of regions of Rna15 that lie outside of the N-terminal RRM responsible for recognition of the polyadenylation signal. We have shown that these regions mediate interactions with the Rna14 and Pcf11 subunits of CF IA that are critical for the function of CF IA in mRNA 3'-end processing. Of particular importance, our analysis of the C-terminal domain of Rna15 and CstF-64 reveals a new all-helical structure that is highly conserved across eukaryotic species. We also find that this domain mediates the interaction of Rna15 with Pcf11, probably through a highly conserved, surface exposed protein/protein interaction patch. Structural changes in the corresponding domain from Pti1 may explain why this very similar protein does not interact with Pcf11 and appears to have a function distinct from that of Rna15. Curiously, the metazoan 3'-end processing machinery has only one Rna15 homologue, CstF-64, whose C-terminal domain sequence is closer to that of Rna15 than Pti1. The biochemical and structural work presented here on these two proteins indicates that the C-terminal domain of CstF64 will also provide a docking platform for Pcf11. Further research will be needed to determine how Pcf11 interacts with Rna15/CstF-64 and with other CF IA subunits and to dissect the implications of these interactions in the communication between transcription termination and 3'-end processing.
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* This work was supported by National Institutes of Health Grants GM041752 (to C. M.) and GM64440 (to G. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables I and II and Figs. 1 and 2.
1 Both authors contributed equally to this work.
2 Present address: Instituto de Química Física Rocasolano. CSIC, Serrano 119, Madrid 28006, Spain.
3 Present address: Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43221.
4 Present address: Dept. of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115.
5 To whom correspondence may be addressed: Dept. of Chemistry, University of Washington, Bagley Hall, Seattle, WA 98195-1700. Tel.: 206-543-7113; Fax: 206-685-8665; E-mail: varani{at}chem.washington.edu.
6 To whom correspondence may be addressed: Dept. of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6935; Fax: 617-636-0337; E-mail: Claire.moore{at}tufts.edu.
7 The abbreviations used are: mRNP, messenger ribonucleoprotein; RNAP, RNA polymerase; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; ChIP, chromatin immunoprecipitation; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; MBP, myelin basic protein; snoRNA, small nucleolar RNA; CTD, C-terminal domain; RRM, RNA recognition motif; CF IA, cleavage factor IA; CPF, cleavage and polyadenylation factor.
8 M. Kim, L. Vasiljeva, O. J. Rando, A. Zhelkovsky, C. Moore, and S. Buratowski, submitted for publication.
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