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First published online February 24, 2006; 10.1104/pp.105.070672 Plant Physiology 140:1507-1521 (2006) © 2006 American Society of Plant Biologists OPEN ACCESS ARTICLE
Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit1,[OA]Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546–0312 (K.J.D., J.Z., K.-Y.Y., D.L.F., A.G.H.); Department of Botany, Miami University, Oxford, Ohio 45056 (R.X., Q.Q.L.); and Department of Biological Sciences, University of Massachusetts, Lowell, Massachusetts 01845 (K.-Y.Y., D.L.F.)
The Arabidopsis (Arabidopsis thaliana) gene that encodes the probable ortholog of the 30-kD subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) is a complex one, encoding small (approximately 28 kD) and large (approximately 68 kD) polypeptides. The small polypeptide (AtCPSF30) corresponds to CPSF30 and is the focus of this study. Recombinant AtCPSF30 was purified from Escherichia coli and found to possess RNA-binding activity. Mutational analysis indicated that an evolutionarily conserved central core of AtCPSF30 is involved in RNA binding, but that RNA binding also requires a short sequence adjacent to the N terminus of the central core. AtCPSF30 was found to bind calmodulin, and calmodulin inhibited the RNA-binding activity of the protein in a calcium-dependent manner. Mutational analysis showed that a small part of the protein, again adjacent to the N terminus of the conserved core, is responsible for calmodulin binding; point mutations in this region abolished both binding to and inhibition of RNA binding by calmodulin. Interestingly, AtCPSF30 was capable of self-interactions. This property also mapped to the central conserved core of the protein. However, calmodulin had no discernible effect on the self-association. These results show that the central portion of AtCPSF30 is involved in a number of important functions, and they raise interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through calmodulin.
Alternative processing of pre-mRNAs is an important factor in sculpting the proteome of eukaryotes, including plants. Thus, in Arabidopsis (Arabidopsis thaliana), numerous instances of alternative splicing have been reported, a phenomenon that may affect as many as 10% of all genes (Kazan, 2003  ; Zhu et al., 2003; Iida et al., 2004). Likewise, many reports of regulated alternative processing have been made (Zhou et al., 2003). The scope of stimuli that have been linked to alternative splicing in plants is extensive, and includes light, environmental stress, development, and challenge by pathogens (Marrs and Walbot, 1997; Hayashi and Nishimura, 1999; Mano et al., 1999, 2000; Isshiki et al., 2000; Jordan et al., 2002; Kong et al., 2003; Staiger et al., 2003). The scope of possible alternative polyadenylation in plants is similarly broad; in one report, it is estimated that as many as 25% of all Arabidopsis genes are alternatively polyadenylated (Meyers et al., 2004). The large size of this estimate likely reflects the pervasive microheterogeneity that exists in the 3'-untranslated regions of plant genes (Dean et al., 1986; Manen and Simon, 1993; Hunt, 1994; Klahre et al., 1995; Rothnie, 1996). However, more dramatic instances of alternative polyadenylation in plants have been described. A well-known example in plants is the FY-mediated control of FCA gene expression, an important determinant of flowering time (Amasino, 2003; Quesada et al., 2003; Simpson et al., 2003).
While many instances of regulated alternative RNA processing in plants have been described, the mechanisms that link stimulus and RNA processing are poorly understood. In mammals, regulated as well as constitutive splicing is controlled to a large extent by so-called SR proteins (Sanford et al., 2003
Regulated polyadenylation in mammals is affected by a number of different mechanisms. Cell cycle control of polyadenylation is accomplished, at least in part, through phosphorylation of poly(A) polymerase, such that the enzyme is phosphorylated and its activity inhibited as the cell enters M phase (Colgan et al., 1996
The Arabidopsis genome possesses genes that are capable of encoding orthologs of virtually the entire suite of known eukaryotic polyadenylation factor subunits (these are summarized at http://www.uky.edu/
Characterization of an Arabidopsis Gene That Encodes a Putative Ortholog of CPSF30
Eukaryotic CPSF30-related proteins contain a distinctive array of CCCH-type zinc-finger motifs (Barabino et al., 1997
The smaller of the two transcripts has a polyadenylation site that lies within an alternatively spliced intron (Fig. 1B) and can encode an approximately 28-kD polypeptide that is similar to eukaryotic CPSF30 proteins. The larger transcript can encode a polypeptide that contains all but the C-terminal 13 amino acids of the 28-kD polypeptide encoded by the smaller transcript, fused to a domain (the so-called YT521-B domain; Stoilov et al., 2002 ) that has been implicated in pre-mRNA splicing in mammals. Nuclear extracts prepared from wild-type Arabidopsis plants contained a number of polypeptides that were recognized by antisera raised against a peptide present in the two At1g30460-derived proteins (WT lane in Fig. 2B). Importantly, two of these (noted with arrowheads in Fig. 2B) were absent in nuclear extracts prepared from the oxt6 insertion mutant (Fig. 2B, compare the oxt6 and WT lanes); the mobilities of these are consistent with those predicted from the properties of the two cDNAs. These mutant plants do not express At1g30460-derived RNAs (oxt6 column in Fig. 2A), indicating that the differences seen in the immunoblot represent the two expected polypeptides. The smaller of these two polypeptides could be immunoprecipitated from nuclear extracts by antibodies raised against another Arabidopsis CPSF subunit, CPSF100 (Elliott et al., 2003), but not by the preimmune serum from the same rabbit (Fig. 2C, compare the antibody and preimmune lanes). It was not possible in this experiment to determine the behavior of the larger At1g30460-encoded polypeptide due to comigration with traces of the immunoglobulin that was present in the immunoprecipitates. In any case, these results indicate that both of the At1g30460-encoded proteins are produced and reside in the nucleus of wild-type Arabidopsis plants, and that the smaller protein resides in a complex that includes another Arabidopsis polyadenylation factor subunit. While the Arabidopsis genome has many other genes that may encode CCCH-like proteins (Fig. 3 ), only the At1g30460-encoded polypeptide(s) resembles CPSF30 to an extent greater than the characteristic Cys/His fingerprint of CCCH proteins. This is revealed in amino acid alignments, which show that At1g30460 falls within a eukaryotic CPSF30 clade to the exclusion of other Arabidopsis CCCH-like proteins (Fig. 3). Sequences closely related to At1g30460 are present in various plant sequence databases (Figs. 1B and 3); interestingly, several expressed sequence tags exist that include both CPSF30- and YT521-B-encoding regions (Fig. 1B), indicating that this protein domain organization is common to plants.
A more detailed comparison of the smaller At1g30460-derived protein with its mammalian and yeast counterparts revealed a high degree of similarity involving three of the five probable zinc fingers that are in the other eukaryotic CPSF30 proteins (Fig. 1A). This similarity was most striking in the second and fourth zinc fingers (using the mammalian protein as a reference). Outside of these three zinc fingers, the similarity between the Arabidopsis and other eukaryotic proteins was minimal. Interestingly, the Arabidopsis protein lacked the first and fifth zinc fingers found in other eukaryotic CPSF30 proteins, as well as the possible CCHC zinc knuckle motifs found in animal CPSF30s (Fig. 1A). Alignment of the full-length plant sequences that can be found in databases reveals additional features that are not apparent in the alignments with other eukaryotic CPSF30 proteins (Fig. 4 ). Thus, while the first zinc-finger motif that is seen in other eukaryotic CPSF30 orthologs is absent in the plant proteins, there is a highly conserved acidic domain at the N termini of the plant proteins (Fig. 4). There is also sequence conservation immediately upstream of the first CCCH motif in the plant proteins, conservation that does not extend to other eukaryotic CPSF30 homologs (compare Figs. 4 and 1A). Downstream from the third (and last) CCCH motif in the plant proteins, there is extensive conservation of a Pro-rich motif; this is the position of the fifth CCCH motif in other eukaryotic CPSF30 proteins. The C termini of the three plant proteins also contain a Gln-rich domain and a highly conserved five-amino acid sequence (PLPQG) near the C terminus (Fig. 4).
RNA Binding by AtCPSF30 Requires the Central Zinc-Finger Domain and an Adjacent Plant-Specific Motif
CPSF30 and Yth1p are both RNA-binding proteins (Barabino et al., 1997
As indicated in Figures 1A and 4, the Arabidopsis CPSF30 contains three conceptual domains: novel plant-specific N and C termini, flanking a conserved central domain defined by the three CCCH-type zinc fingers of the Arabidopsis protein. To better understand the roles of these domains (N, Z, and C, respectively) in the RNA-binding activity of the protein, a series of deletion derivatives was prepared and assayed for RNA binding. Deletion of the entire N terminus (up to the first of the three zinc-finger motifs) dramatically reduced the RNA-binding activity of AtCPSF30 (m5 in Fig. 5B). Thus, at protein concentrations at which maximal binding is seen with the wild-type protein (2–5 µM), the N-terminal deletion displayed little binding. Some binding could be discerned at concentrations above 5 µM; it was not possible to add enough protein to reach saturation in these assays, but the apparent affinity of the RNA for protein in this assay was at least 20-fold less than that seen with the wild-type protein (data not shown). In contrast, deletion of the C terminus had little discernible effect on RNA binding (m4 in Fig. 5B). In particular, there was little difference between the C-terminal truncation and the full-sized protein in terms of the profile of binding activity as a function of protein concentration, with activity decreasing precipitously at concentrations below 1 µM. As expected (based on the properties of the N-terminal deletion), the central domain by itself did not bind RNA (m2 in Fig. 5B; those mutants that displayed no detectable binding are listed in Fig. 5B). Moreover, the isolated C-terminal domain was also unable to bind RNA (m8 in Fig. 5B). Elimination of the second and third zinc-finger motifs from the m4 mutant eliminated RNA binding as well (m1 in Fig. 5B). The plant-specific N domain of AtCPSF30 consists of two evolutionarily conserved sequences: an acidic domain at the very N terminus of the protein and a conserved region that abuts the first zinc finger (Fig. 4). The effects on RNA binding of deletion of the N terminus in the m5 mutant might be attributable to elimination of either (or both) of these sequences. To explore the possibilities, the 11 amino acids that are adjacent to the first zinc finger were added to the Z + C and Z proteins (m9 and m10, respectively, in Fig. 5B). In contrast to the m5 protein, RNA binding was readily discernible with the m9 mutant (Fig. 5B). Interestingly, the apparent affinity of RNA for this protein was about 5-fold lower than the wild-type or m4 proteins. While the Z domain by itself (m2) was unable to bind RNA, the m10 mutant displayed considerable RNA-binding activity (Fig. 5B). The apparent affinity of RNA for this protein was some 3- to 5-fold lower than that for the wild-type and m4 proteins, comparable with that of the m9 protein. Based on these results, it can be concluded that RNA binding by AtCPSF30 requires the central zinc-finger domain and the plant-specific sequence immediately adjacent to the first zinc-finger motif.
Among the computer-assisted analyses of the polypeptide encoded by At1g30460 that were performed was a search for calmodulin-binding motifs. This yielded a possible domain (underlined in Fig. 1A) that abutted the N-terminal zinc-finger motif. Accordingly, the ability of this protein to bind calmodulin was examined using a calmodulin blotting assay. As shown in Figure 6 , the full-sized MBP-AtCPSF30 protein as well as a predominant breakdown product displayed calmodulin-binding activity in this assay (lanes marked "WT"). In contrast, comparable quantities of purified MBP lacked calmodulin-binding activity (lanes marked "MBP"). This indicates that the AtCPSF30 portion of the fusion protein is responsible for the observed calmodulin binding.
To test the hypothesis that this calmodulin binding was due to the domain identified in the computational analysis, a mutational approach was taken. Initial studies involved deletion analysis of the protein, examining the abilities of different parts of the protein to bind calmodulin in a far-western assay. For these studies, the three domains of AtCPSF30 were produced in various combinations as MBP fusion proteins. None of the isolated domains (N, Z, and C, respectively) bound calmodulin in this assay (m7 in Fig. 6; data not shown). However, the N + Z construct (m1 in Fig. 6) did bind calmodulin. This suggested that the calmodulin-binding domain might be located near the breakpoints between the N and Z domains, which is consistent with the computer prediction. To confirm this, four point mutations were introduced into the full-sized AtCPSF30 coding sequence, as indicated in Figure 6 ("30M"). In contrast to the full-sized protein, the three independent mutant proteins did not bind calmodulin (Fig. 6, lanes labeled "30M-4," "30M-5," and "30M-6"). From these results, we conclude that the calmodulin-binding domain of AtCPSF30 is situated immediately upstream of the first conserved zinc finger of the protein, in a region that itself is highly conserved in plants (Fig. 4). The near juxtaposition of the calmodulin-binding and RNA-binding domains in AtCPSF30 suggests that calmodulin may affect the ability of AtCPSF30 to bind RNA. This prediction was tested by examining the effects of calmodulin on RNA binding by AtCPSF30. In the presence of calcium, calmodulin inhibited the RNA-binding activity of AtCPSF30 by approximately 80% ("WT + Ca" sample in Fig. 7 ). Replacement of calcium chloride with EGTA eliminated the inhibition ("WT + EGTA" sample in Fig. 7).
As further confirmation that calmodulin inhibits RNA binding by AtCPSF30, similar studies were performed with the 30M mutant. As shown in Figure 5B, this mutant displayed an RNA-binding activity that was similar to that of the wild-type protein and the m4 mutant. This indicates that the four Ala substitutions in the 30M mutant do not affect the RNA-binding activity of the protein, even though they are within the region that is needed for RNA-binding activity. To test the hypothesis that the identified calmodulin-binding domain was responsible for the inhibitory effects of calmodulin on RNA binding, the effects of calmodulin on the RNA-binding activity of the mutant were examined. In contrast to the behavior of the wild-type AtCPSF30, the 30M mutant was not affected by calmodulin in the presence of calcium ("MUT + Ca" sample in Fig. 7). RNA binding was not affected by calmodulin when calcium chloride was replaced with EGTA ("MUT + EGTA" sample in Fig. 7). This is consistent with the observation that the 30M mutant does not bind calmodulin and indicates that the conserved sequence immediately preceding the first zinc-finger motif is necessary for the inhibition of RNA binding by calmodulin.
The curve for binding of AtCPSF30 with RNA (Fig. 5B) has characteristics of strong cooperativity. This in turn suggests that AtCPSF30 is capable of interacting with itself. This hypothesis was tested by copurification assays. For this, a series of labeled AtCPSF30 derivatives (illustrated in Fig. 8A ) was prepared and used in copurification assays using MBP-AtCPSF30 as bait. As shown in Figure 8B, when AtCPSF30 ("wt") was incubated with purified MBP-CPSF30 and the MBP fusion protein was subsequently purified using amylose resin, there was a significant copurification of the labeled protein (top; lane labeled "wt"). Replacement of the MBP-CPSF30 with purified MBP eliminated this copurification (middle). This result indicates that AtCPSF30 interacts with itself.
To define the domain of AtCPSF30 that mediates the self-association, similar experiments were done with labeled portions of AtCPSF30. As shown in the top section of Figure 8B, no apparent copurification of the plant-specific N and C termini of AtCPSF30 (m6, m7, and m8, respectively) with MBP-AtCPSF30 could be seen in the copurification assay. In contrast, significant quantities of the evolutionarily conserved central domain (m2) did copurify with MBP-AtCPSF30. Replacement of MBP-AtCPSF30 with MBP eliminated this copurification (middle section of Fig. 8B). These results indicate that the central domain of AtCPSF30 is the part of the protein responsible for the self-association of this protein. The cooperativity that is suggested by the binding curve (Fig. 5) and direct demonstration of protein-protein interaction (Fig. 8B) suggests a possible mechanism for the inhibition by calmodulin of RNA binding by AtCPSF30. Specifically, it is possible that calmodulin inhibits the self-interaction of AtCPSF30, thus eliminating the possibility of cooperative binding of the protein to RNA. To test this, the effects of a large molar excess of calmodulin on the self-association were examined. As shown in Figure 8C, calmodulin had no discernible effect on the copurification of labeled AtCPSF30 with MBP-AtCPSF30. This result suggests that calmodulin does not affect the self-association and, thus, that the inhibition of RNA binding by calmodulin is not mediated by an inhibition of self-association of AtCPSF30.
A Functional Map of a Putative Plant Polyadenylation Factor Subunit
The results presented in this study permit the construction of a functional map (Fig. 9
) of AtCPSF30, the plant ortholog of the 30-kD subunit of the CPSF. Specifically, RNA binding by this protein requires much of the central zinc-finger domain that is conserved in all eukaryotic CPSF30 orthologs (Fig. 1A), as well as some 11 amino acids immediately next to the N terminus of the three zinc-finger motifs. This latter domain is conserved in plant CPSF30-like proteins but not in its eukaryotic counterparts (Figs. 1A and 4). Calmodulin binding involves the same plant-specific domain that is adjacent to the first zinc-finger motif. However, the observation that the m7 mutant is impaired in calmodulin binding (Fig. 6), even though it contains this motif, indicates that some sequences within the central zinc-finger domain are also needed for the interaction with calmodulin. While there is some overlap in the calmodulin- and RNA-binding domains, these functions can be separated; thus, the four Ala substitutions in the 30M mutant affect calmodulin binding, but not RNA binding itself. This suggests that these two functions involve different amino acids. It also provides a model for the inhibition of RNA binding by calmodulin; specifically, it suggests that calmodulin and RNA occupy the same surface of AtCPSF30, such that occupancy by calmodulin prevents access to the protein by RNA. A somewhat similar mechanism has been suggested for the inhibition by calmodulin of DNA binding by the E-protein family of mammalian basic helix-loop-helix proteins (Onions et al., 1997
The third function that has been mapped in this study is the self-association of AtCPSF30. This activity can also be attributed to the central zinc-finger domain of the protein. However, the apparently cooperative nature of binding of RNA by AtCPSF30 suggests that RNA binding and self-association are not mutually exclusive; indeed, they may be reinforcing. Additionally, while calmodulin binding involves at least part of the zinc-finger domain, it does not seem to have an impact on the self-association of AtCPSF30. Thus, while calmodulin- and RNA-binding are mutually exclusive activities of AtCPSF30, calmodulin binding and the self-association are not. These considerations permit a tentative demarcation of the central zinc-finger domain, with the N-terminal portion being involved in RNA and calmodulin binding, and the C terminal part probably important for self-association. That self-association involves one or more zinc-finger motifs is consistent with the frequent association of zinc-finger domains with protein-protein interactions (Matthews and Sunde, 2002
AtCPSF30 is the only Arabidopsis protein with a degree of sequence similarity to other eukaryotic CPSF30 proteins that extends beyond the typical spacing of Cys and His residues in the CCCH zinc-finger motif, and a number of lines of evidence support the conclusion that AtCPSF30 is an authentic polyadenylation factor subunit. As is the case with its yeast counterpart (Yth1p; Barabino et al., 1997 In yeast, the gene that encodes its CPSF30 ortholog, YTH1, is essential. In contrast, the At1g30460 gene in Arabidopsis is not absolutely essential for the growth of the plant, as adult, fertile plants that bear a T-DNA insertion within the first exon can be obtained. While it is not apparent from similarity searches of Arabidopsis databases, it is nonetheless possible that other distantly related proteins (perhaps one or more of the family of CCCH motif-containing proteins listed in Fig. 3) may serve analogous roles in mRNA 3'-end formation in Arabidopsis. Alternatively, AtCPSF30 may function in a somewhat different fashion in mRNA 3'-end formation than its yeast counterpart, serving a nonessential role. The central zinc-finger domain of AtCPSF30 is also involved in the self-association of this protein (Fig. 8B). There have been no reports of self-association of CPSF30 in other systems. Whether this reflects a difference between plants and other eukaryotes is an open issue. Nonetheless, the self-association described in this report raises interesting possibilities with respect to mRNA 3'-end formation in plants and to the role(s) that AtCPSF30 may play in RNA processing and metabolism. For example, RNA recognition by a multimer (such as a dimer) would permit AtCPSF30 to recognize more than one sequence element in a plant polyadenylation signal.
The At1g30460 gene encodes two polypeptides, AtCPSF30 and a larger polypeptide that contains all but the last 13 amino acids of AtCPSF30 fused to the N terminus of another domain (Fig. 1A). The latter includes a motif (the so-called YT521-B motif) that was first described in proteins that are associated with pre-mRNA splicing in mammals (Stoilov et al., 2002 The potential commonalities or links between splicing and polyadenylation extend beyond the presence in plants of a protein with polyadenylation- and splicing-related functionalities. Given that the central domain of AtCPSF30 is capable of interacting with itself (Fig. 8), it follows that the larger At1g30460-encoded protein is expected to be capable of interactions with itself and with AtCPSF30. Thus, three different classes of multimeric complexes containing the AtCPSF30 polypeptide can be formed in the cell. These different variants have the possibility of acting in somewhat different ways; complexes consisting of just AtCPSF30 may act in polyadenylation, while complexes containing the AtCPSF30-YT521-B polypeptide may work in splicing. The possibility also exists that heteromeric AtCPSF30-containing complexes may mediate communication between splicing and polyadenylation. These considerations provide a number of potential conceptual links between pre-mRNA splicing and polyadenylation in plants. Further biochemical characterization as well as studies of mutants bearing defects in the At1g30460 gene promise to shed interesting new insight into the interplay between different RNA-processing events in plants.
Finally, the effects of calmodulin on RNA binding by AtCPSF30 are of interest for several reasons. In animals, CPSF30 is a target of the influenza virus-encoded protein NS1; binding of this protein to CPSF30 inhibits polyadenylation, thereby contributing to the shutdown of host gene expression in virus-infected cells (Nemeroff et al., 1998
In conclusion, these studies demonstrate that an Arabidopsis protein (AtCPSF30) that is related to the eukaryotic polyadenylation factor subunit CPSF30 is an RNA-binding protein. This protein also interacts with calmodulin such that, in the presence of calmodulin and calcium, its RNA-binding activity is inhibited. AtCPSF30 also interacts with itself, raising the possibility that it acts in RNA processing as a multimer. These results indicate that RNA processing in plants is probably regulated by stimuli that signal through calmodulin.
Sequence Analysis
Sequence data were analyzed using Vector NTI software (Informax). Multiple amino acid sequence alignments were performed using ClustalX 1.83 and unrooted trees generated using the same software package. Trees were drawn using TreeView version 1.6.2. Calmodulin-binding predictions were performed using the service at http://calcium.uhnres.utoronto.ca/ctdb/ctdb/sequence.html (Yap et al., 2000
The oxt6 mutant, bearing a T-DNA insertion in the first exon of At1g30460, was identified in a collection of Arabidopsis (Arabidopsis thaliana) Columbia that had been mutagenized by T-DNA (pROK2) insertion; this collection was obtained from the Arabidopsis Biological Resource Center (Ohio State University). The position of the insertion and genotype of the plants were confirmed by PCR and RNA-blot analyses. This mutant is viable but has a somewhat dwarfed growth stature. Details of the isolation of the mutant and its characteristics will be published elsewhere.
RNA-blot analysis was conducted as described previously (Xu et al., 2004
Arabidopsis Nuclei Isolation and Nuclear Protein Extraction Leaves of 3- to 4-week-old plants were used for nuclei isolation. Leaves were frozen and ground in liquid nitrogen to fine powder with a mortar and pestle. All subsequent steps were carried out on ice or at 4°C. About 20 g of tissue powder were mixed with 100 mL of extraction buffer (250 mm Suc, 10 mm PIPES-KOH, pH 7.0, 10 mm KCl, 10 mm MgCl2, 0.3% Triton X-100, 1 mm dithiothreitol, and 0.2 mm phenylmethylsulfonyl fluoride) on ice for 30 min, then filtered through two layers of Miracloth and subjected to centrifugation at 2,000g for 10 min. The pellet was resuspended in 15 mL of nuclei washing buffer (NWB; 0.4 M hexylene glycol, 10 mM PIPES-KOH, pH 7.0, 10 mM MgCl2, 0.3% Triton X-100), filtered through a 100-µm nylon mesh, then layered on 15 mL of 30% Percoll in NWB and centrifuged again at 1,000g for 30 min. The pellet was resuspended in 20 mL of NWB and filtered through a 100-µm nylon mesh. Nuclei were further purified in a discontinuous Percoll gradient made by layering 10 mL of 30% Percoll in NWB on 10 mL of floating buffer (0.44 M Suc, 80% Percoll, 10 mM PIPES-KOH, pH 7.0, 10 mM MgCl2). The gradient was centrifuged at 200g for 5 min. Most of the nuclei formed a layer just above floating buffer. They were removed, washed once with NWB (without addition of Triton X-100), and finally collected by centrifugation. This nuclei-enriched fraction was resuspended in protein sample buffer (0.175 M Tris-HCl, pH 8.8, 5% SDS, 15% glycerol, 30 mM dithiothreitol) subjecting to SDS-PAGE gels for western-blot analysis. Nuclear protein extract used for coimmunoprecipitation was prepared using buffer X (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 150 mM NaCl, 0.2 mM EDTA, 0.5% Triton X-100, 25% glycerol, protease inhibitor cocktail [Sigma]) and treated twice with sonication (5 s each), followed by an incubation on ice for 4 h and centrifugation. Protein concentration was determined using the Bradford reagent (Sigma).
Antibodies were prepared against synthetic peptides (RFFRLYGECREQDC for AtCPSF30 and YNHRKERHLNGTVLC for AtCPSF100) from the predicted protein sequences of AtCPSF30 and AtCPSF100 (with an additional C each for conjugation purpose). After conjugation with KLH carrier protein using the Imject maleimide activated mcKLH kit (Pierce), the conjugant was injected into two rabbits (200 µg/injection). Each rabbit was given four booster injections about 2 weeks apart. Peptide-specific antibodies were purified using an affinity-purification procedure as described (Li et al., 1998
For coimmunoprecipitation experiments, 20 µL of affinity-purified antibody was added to 200 µg of nuclear protein in 300 µL of PBST buffer (0.14 M NaCl, 0.008 M sodium phosphate, 0.002 M potassium phosphate, 0.01 M KCl, pH 7.4, 0.5% Triton X-100) and incubated overnight with gentle shaking at 4°C. Forty microliters of Affi-Gel protein A (Bio-Rad) was then added and incubated for 2 to 4 h at 4°C. The beads were collected by centrifugation and washed six times with 400 µL of PBST buffer. The affinity-bound proteins were eluted from the beads by boiling for 5 min in a SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.7 M
DNAs encoding the AtCPSF30 coding region were isolated from total RNA by RT-PCR as described elsewhere (Addepalli et al., 2004 Deletion derivatives of AtCPSF30 were amplified by PCR using the full-length AtCPSF30 cDNA clone as a template and the primers listed in Table I. The sequence of cloning into pGEM, sequencing, and cloning into pMALC2 described in the preceding paragraph was followed for each clone. The calmodulin-insensitive AtCPSF30 mutant was generated from the pMALC2-AtCPSF30 clone using the QuickChange XL site-directed mutagenesis kit (Stratagene) and the oligonucleotides indicated in Table I; several independent clones were isolated, the mutation confirmed by DNA sequencing, and further analysis performed as indicated in the following.
To produce MBP fusion proteins, 200 mL of LB media were inoculated with 10 mL of an overnight culture of transformed Rosetta (D3) cells and the 200-mL cultures grown at 37°C for 3 to 4 h. Expression of the fusion protein gene was induced by addition of 200 µL of 1 M isopropylthio-
For RNA binding, the electrophoretic mobility shift assays described elsewhere (Das Gupta et al., 1998 For the experiment shown in Figure 7, RNA-binding reactions were supplemented with CaCl2 (10 µM) or EGTA (1 mM), or purified Arabidopsis calmodulin (Cam6, the product of the Arabidopsis gene At5g21274, provided by Dr. Ray Zielinski, University of Illinois).
The binding of recombinant proteins to calmodulin was assessed using the Stratagene Affinity CBP detection system (catalog no. 200370). In some cases, biotinylated calmodulin from STI Signal Transduction Products was used in place of the same reagent from Stratagene.
DNA fragments encoding various parts of AtCPSF30 were cloned into pCITE (Novagen) as the same BglII fragments used to clone them into pMALC2. Recombinant pCITE plasmids were used to program in vitro translation reactions using the STP3 (Novagen) kit, exactly as recommended by the manufacturer. (Note that this protocol includes a postreaction treatment with RNAse A.) Five microliters of the translation reactions were added to 1 µL of MBP or MBP-AtCPSF30 (containing approximately 1 µg of purified protein, in GLB). After 30 min at 30°C, the reactions were added to a mixture containing 25 µL (packed resin volume) of amylose resin (New England Biolabs) in 100 µL of GLB + 0.1% Nonidet P-40. After 5 min of gentle rocking at room temperature, the resin was collected by centrifugation (5 s in a microcentrifuge), washed three times with GLB + 0.1% Nonidet P-40, and suspended in 25 µL of SDS-PAGE sample buffer. After boiling for 5 min, 10-µL aliquots were separated by SDS-PAGE, and the gels were dried and analyzed by autoradiography using a phosphorimager. For the experiment shown in Figure 7C, calmodulin (1 µg), CaCl2 (final concentration of 3 mM), and EGTA (final concentration of 3 mM) were added in additional volumes of 3 µL total. The volumes of the controls in this experiment were adjusted accordingly. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AY140901.
We thank Carol Von Lanken for technical assistance, and Dr. Balasubrahmanyam Addepalli and Dr. Ray Zielinski for gifts of MBP and calmodulin, respectively. Received September 29, 2005; returned for revision December 20, 2005; accepted January 27, 2006.
1 This work was supported by the National Science Foundation (grant no. MCB–0313472 to A.G.H. and Q.Q.L.), the U.S. Department of Agriculture National Research Initiative (grant no. 2001–00911 to D.L.F.), and the Kentucky Tobacco Research and Development Center (D.L.F.). 
2 These authors contributed equally to the paper.
3 Present address: Department of Surgery, University of Wisconsin-Madison, 600 Highland Ave., Madison, WI 53792. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Arthur G. Hunt (aghunt00{at}uky.edu).
[OA] Open Access articles can be viewed online without a subscription. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.070672. * Corresponding author; e-mail aghunt00{at}uky.edu; fax 859–257–7125.
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-mercaptoethanol, 0.1% bromphenol blue) before loading onto 10% SDS-PAGE for electrophoresis. 
