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. 2011 Sep 9;157(3):1546–1554. doi: 10.1104/pp.111.179465

A Novel Plant in Vitro Assay System for Pre-mRNA Cleavage during 3′-End Formation1,[OA]

Hongwei Zhao 1,2,3, Jun Zheng 1,2, Qingshun Quinn Li 1,*
PMCID: PMC3252153  PMID: 21908687

Abstract

Messenger RNA (mRNA) maturation in eukaryotic cells requires the formation of the 3′ end, which includes two tightly coupled steps: the committing cleavage reaction that requires both correct cis-element signals and cleavage complex formation, and the polyadenylation step that adds a polyadenosine [poly(A)] tract to the newly generated 3′ end. An in vitro biochemical assay plays a critical role in studying this process. The lack of such an assay system in plants hampered the study of plant mRNA 3′-end formation for the last two decades. To address this, we have now established and characterized a plant in vitro cleavage assay system, in which nuclear protein extracts from Arabidopsis (Arabidopsis thaliana) suspension cell cultures can accurately cleave different pre-mRNAs at expected in vivo authenticated poly(A) sites. The specific activity is dependent on appropriate cis-elements on the substrate RNA. When complemented by yeast (Saccharomyces cerevisiae) poly(A) polymerase, about 150-nucleotide poly(A) tracts were added specifically to the newly cleaved 3′ ends in a cooperative manner. The reconstituted polyadenylation reaction is indicative that authentic cleavage products were generated. Our results not only provide a novel plant pre-mRNA cleavage assay system, but also suggest a cross-kingdom functional complementation of yeast poly(A) polymerase in a plant system.

Gene expression in eukaryotes requires the transcription of DNA into mRNA in the nucleus. The newly transcribed pre-mRNAs undergo extensive processing, such as 5′-end capping and removal of introns and 3′-end polyadenylation before they are ready to be transported to the cytoplasm for translation. Among pre-mRNA processing events, 3′-end formation and polyadenylation are known to regulate transcription termination, affect intron splicing, promote mRNA transportation and translation initiation, and protect mature mRNAs from unregulated degradation (Buratowski, 2005; Moore and Proudfoot, 2009). In addition, studies on alternative polyadenylation show that more than half of plant and mammal genes can be alternatively processed at different locations, resulting in distinct mature mRNAs from the same pre-mRNA transcripts (Tian et al., 2005; Xing and Li, 2010; Wu et al., 2011). More recently, the choice of alternative polyadenylation sites at 3′-untranslated region (UTR) of many genes has been linked to gene expression level control and cancer development (Mayr and Bartel, 2009). Thus, a theme of gene expression regulation through pre-mRNA polyadenylation is emerging. Due to its tight connections to transcription, translation, and RNA decay, mRNA 3′-end polyadenylation appears to act as a hub for fine tuning gene expression and forming a regulation network (Danckwardt et al., 2008).

The 3′-end processing of mRNA includes two coupled steps: cleavage at a specific site at the 3′-UTR and an addition of a polyadenosine [poly(A)] tract to the newly formed 3′ end. The biochemical process of polyadenylation is relatively well studied in mammals and yeast (Saccharomyces cerevisiae). The 3′-end cleavage and polyadenylation reaction is guided by sequence elements (cis-elements) in pre-mRNA, which are recognized by a protein complex that carries out enzymatic cleavage and polyadenylation reaction at a specific site. This apparently simple process directly involves several cis-elements, as well as more than 14 protein factors in mammals (Zhao et al., 1999; Mandel et al., 2008). These 14 protein factors can be divided into several subcomplexes, such as cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor, cleavage factor I, and cleavage factor II. Meanwhile, poly(A) polymerase (PAP), poly(A) binding protein-II, symplekin, and C-terminal domain of the RNA polymerase II have also been found to be necessary in this 3′-end processing complex (Mandel et al., 2008). In yeast, a similar set of proteins, albeit named differently, were also identified to be important for 3′-end processing (Mandel et al., 2008). A recent report revealed the involvement of over 80 proteins that form a complex with functional poly(A) signals (Shi et al., 2009). The functionality of this larger collection of associated proteins in polyadenylation, however, remains to be confirmed.

When investigating the mechanisms of mRNA 3′-end formation in mammal and yeast, in vitro assay systems were found to be critical in identifying and characterizing components of the cleavage and polyadenylation complex (Zhao et al., 1999). These in vitro assay systems simplified the isolation and identification of new protein factors involved in this process, making it much easier to perform subsequent functional studies and test the functions of cis-elements. Until now, however, an in vitro assay system that can accurately cleave and polyadenylate pre-mRNA in plants has never been established, to our knowledge, and the lack of such a system has affected the study of 3′-polyadenylation machinery in plants (Rothnie, 1996; Li and Hunt, 1997). To address this, numerous efforts were made in establishing a plant cleavage and polyadenylation in vitro assay system by using the nuclear extracts of different plants, such as peas (Pisum sativum), cauliflower (Brassica oleracea), and Arabidopsis (Arabidopsis thaliana), but without success. Over the past few years, through bioinformatics, phylogenetics, protein interactions, and proteomic analysis, we and others have identified the conserved polyadenylation factors (Simpson et al., 2003; Herr et al., 2006; Hunt et al., 2008; Zhao et al., 2009). However, the biochemical efficacy of these proteins remains largely elusive. Here, we report a plant in vitro mRNA 3′-end assay system that cleaves pre-mRNA at specific sites as found in vivo. Nuclear protein extracts from Arabidopsis suspension cells have been shown to carry out the committing cleavage step. When complemented with a yeast PAP (yPAP), a full cleavage and polyadenylation activity was reconstituted. This assay system will be useful for studying plant mRNA 3′-end formation mechanisms as well as other related RNA processing events.

RESULTS

Establishment of a Plant in Vitro Pre-mRNA Cleavage System Using Soluble Nuclear Proteins from Arabidopsis Suspension Cells

While studying the proteomics of plant polyadenylation factors, we overexpressed Arabidopsis CPSF73-I (for CPSF 73-kD subunit-I; At1g61010) using a tandem affinity purification tagging approach and isolated the protein complexes formed with it (Zhao et al., 2009). Arabidopsis CPSF73-I is a homolog of mammalian CPSF73 that was shown to be the endonuclease that plays a direct role in the cleavage step of pre-mRNA 3′-end formation (Dominski et al., 2005; Mandel et al., 2006). Thus, it was reasonable to examine whether AtCPSF73-I plays the same role of cleaving pre-mRNAs in plants. To accomplish this, the soluble nuclear proteins from Arabidopsis suspension cells overexpressing AtCPSF73-I were prepared from nuclei. This extract was free of organelle contamination such as chloroplast (Fig. 1A), and was used for the initial establishment of the assay conditions.

Figure 1.

Figure 1.

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RNA substrate and endonuclease cleavage activity of nuclear extracts from Arabidopsis culture cells. A, Examination of nuclear extracts for chloroplast contamination. Left section, Coomassie-Blue-stained SDS-polyacrylamide gel loaded with equal amounts of protein from whole-cell extracts (WCE-CI), and nuclear extracts (Nu-CI) from culture cells overexpressing AtCPSF73-I (or CI). Right section, Proteins from a replicate gel were transferred to a membrane and probed for the chloroplast stromal Rubisco large subunit (RbcL, approximately 45 kD) using a specific antibody (Agrisera). Protein size markers (kD) are indicated on the right. B, A schematic representation of the CaMV STS pre-mRNA substrate used in this study. The 3′-UTR region includes the FUE, the NUE, and the CE (including cleavage sites [CS], indicated by arrows), with the size of cleavage products indicated (not drawn to scale). The sizes of nucleotide deletions on the FUE and NUE for mutational studies are also indicated under these elements. CDS, Coding sequence. C, Detection of cleavage activity with uniformly labeled STS and reverse STS, with or without Nu-CI. D, Cleavage of 5′-end-labeled STS using Nu-CI. The arrow indicates the expected 182-nt cleavage product. E, Cleavage of 3′-end-labeled STS by Nu-CI. The arrow indicates the expected 20-nt 3′-end product. F, Cleavage of uniformly labeled STS pre-mRNA by nuclear protein extracts from wild-type cells (Nu-WT). The arrow indicates the expected 182-nt 5′-end product. In D to F, the control in each section refers to reactions incubated with buffer in place of the nuclear extract. In C to F, substrates and cleavage sites (marked by black triangles), and the way the RNA substrates were labeled (either uniform or end labeled; marked by stars) are indicated at the left-hand side; RNA size standards (nt) are indicated on the right-hand side.

A 3′-UTR from the pre-mRNA of cauliflower mosaic virus (CaMV) transcript clone named STS was used as the substrate for this in vitro cleavage assay system. The cleavage and polyadenylation profile of STS has been extensively studied (Mogen et al., 1990; Rothnie et al., 1994). Based on conventional genetic studies, it has been demonstrated that STS is 200 nucleotides (nts) in length and contains three well-defined cis-elements: the far-upstream element (FUE), the near-upstream element (NUE), and a cleavage element (CE) around the cleavage/polyadenylation site (Fig. 1B). Accordingly, the nuclear extract was tested for its cleavage activity using uniformly [-32P] ATP-labeled STS in assay conditions as described in the “Materials and Methods” section. To our surprise, the nuclear proteins could cleave STS into RNA about 182 nt in length (Fig. 1C, lane 3), the expected size range as revealed by in vivo assays (Rothnie et al., 1994). This cleavage only occurred on the sense transcripts of STS since the reverse (or antisense) transcript of it could not be cleaved (compare lanes 1 and 3 in Fig. 1C), indicating the observed cleavage is sequence specific. To confirm that the observed cleavage is at the 3′ end, where the in vivo cleavage occurs, a 5′-end 32P-labeled STS was generated and subjected to the same assay conditions. Again, the cleavage reaction left a product that was about 182 nts in length (Fig. 1D, the middle lane). STS was also 3′-end labeled by [5′-32P] pCp, using T4 RNA ligase, and then subjected to the same assay. As shown in Figure 1E (lane 1), a 3′-end cleavage product of about 20 nts was detected. To further confirm the cleavage site at the sequence level, the 182-nt band was cloned and sequenced. In agreement with the in vivo experiments, the results showed that the cleavage occurred at the same two sites observed by Rothnie et al. (1994) producing two different lengths, 180 and 182 nt (not seen as two bands on the gel possibly due to resolution; Rothnie et al., 1994). These results indicate that the cleavage was carried out by an endonuclease generating both the 5′ and 3′ cleavage products. This excludes the possibility that such products were generated by an exonuclease activity. Thus, an in vitro assay system for pre-mRNA cleavage was established by using the nuclear proteins from cells overexpressing AtCPSF73-I.

Since we found cleavage activity based on nuclear extracts overexpressing AtCPSF73-I in the wild-type cell background, we further asked if the overexpression of AtCPSF73-I was required for this activity. To address this question, we generated nuclear extracts from nontransformed, wild-type culture cells, and tested for their cleavage activity. Surprisingly, the wild-type nuclear protein was also able to correctly cleave the pre-mRNA (Fig. 1F). Therefore, our results indicated that specific pre-mRNA cleavage activity can be found in both the AtCPSF73-I overexpressing and the wild-type nuclear protein extracts. Although the overexpressed materials helped us in the establishment of the assay system, overexpression of the plant polyadenylation factors was not necessary for this activity. So some of the following experiments were done using nuclear protein extracts from AtCPSF73-I overexpressing cells (called Nu-CI). All important key experiments were repeated using wild-type nuclear extracts (called Nu-WT), some of which are shown as indicated.

The in Vitro Cleavage System Is cis-Element Dependent

To demonstrate that the accurate cleavage can also happen to other transcripts than STS, we generated a few transcripts with downstream extensions on the 3′-UTR of the native CaMV STS, but without changing the poly(A) signals. The results showed that the cleavage sites were maintained, despite that the pre-RNA molecules extended to different lengths at their 3′ ends (Fig. 2A). This result indicates that the observed cleavage depends on cis-elements embedded in the sequence of the substrate upstream of the cleavage site. The accuracy of cleavage is not affected by sequence downstream of the cleavage site. These results agree with Rothnie and Mogen’s in vivo observation, which showed both FUE and NUE but not other downstream elements contribute to pre-mRNA cleavage site selection (Mogen et al., 1990; Rothnie et al., 1994). To dissect the potential cis-elements [poly(A) signals] located in the upstream of the pre-mRNA, different mutations of the STS pre-mRNA were employed. These mutants were designed based on previous studies on published in vivo studies where a disruption at either the FUE or the NUE region caused utilization of cryptic polyadenylation sites (Mogen et al., 1990; Rothnie et al., 1994). Similar observations were made in our in vitro experiments where a deletion of FUE (22 nts) resulted in cleavage at an alternative site upstream of the original site (Fig. 2B). Deletion of NUE (6 nts) disrupted the cleavage at the correct sites, but resulted in the use of nonspecific sites downstream of the original cleavage sites (Fig. 2C). Such results can be typically found in genetic studies where the utilities of alternative sites were reflected by read through the length of detection probes or cryptic sites (Mogen et al., 1990; Rothnie et al., 1994). These results indicate that accurate cleavage requires correct poly(A) signals, as it was revealed by previous studies.

Figure 2.

Figure 2.

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Properties of the cleavage activity. RNA substrates and cleavage sites are indicated at the left-hand side of gels. A, Cleavage of STS and its 3′-end extension variants. The total lengths are the STS plus the indicated numbers of nts on the top of the lanes. Note that all the STSs were cleaved at the same site, leaving the same 182-nt 5′-end products as indicated by arrowheads. Reactions were incubated with nuclear extracts (Nu-CI; +), and without nuclear extracts added (−). B and C, Cleavage reactions require both FUE and NUE cis-elements. Internal deletions of 22 nts of the FUE element from STS (B), or 6 nts of the NUE element from STS58 (C; 58-nt 3′-end extension of STS) abolish formation of site-specific cleavage products. The locations of expected cleavage products are marked by arrows. Control reactions were incubations without nuclear extract addition. D, Specific cleavage products of the native Arabidopsis Rubisco small subunit (rbcS) transcript (At5g38420) 3′-UTR by Nu-CI. Cleavage products are indicated by white triangles (sizes labeled by asterisk [*]). All substrate RNAs were uniformly labeled. RNA size markers are in nts.

The in Vitro Cleavage System Functions on Native Transcripts

Furthermore, we tested if the cleavage activity also works on endogenous Arabidopsis genes, besides the viral one. A representative 3′-UTR of a Rubisco small subunit gene (At5g38420) was chosen due to its high expression level and abundance of available ESTs. When this pre-mRNA was processed in vitro using our cleavage assay prepared from Nu-CI, two bands (Fig. 2D) representing two groups of poly(A) sites were detected. Sequencing results showed these two bands corresponded to two authentic poly(A) sites supported by ESTs that have poly(A) tails attached to them. These EST data are from the 8K poly(A) site dataset as described previously by Loke and coworkers (2005), the National Center for Biotechnology Information Arabidopsis EST collection (http://www.ncbi.nlm.nih.gov), and the Arabidopsis Information Resources databases (www.arabidopsis.org). Specifically, the two poly(A) site groups were found at the 3′-UTR of At5g38420 with TAIR9 genome coordinates 15381033 to 15381036 (for band 171–174 in Fig. 2D), and 15380980 to 15380988 (for the band marked 219–227 in Fig. 2D). Similar results were also obtained with nuclear extract prepared from wild-type Arabidopsis cell cultures (data not shown). Taken together, the cleavage activity we found is an authentic mRNA 3′-end processing component functioning on both a viral RNA in a poly(A) signal-dependent manner and native plant pre-mRNA transcripts.

Optimization of the Cleavage Reaction and Cleavage Kinetics

To optimize the conditions for the cleavage assay, different temperatures, pH gradients, and reaction time were tested. The highest cleavage efficiency was observed at 30°C, where more than 95% of pre-mRNA was cleaved (Fig. 3A). The most efficient cleavage was found at pH 8.5 (Fig. 3B), but it remained highly active from pH 8.0 to pH 10.0. When a time course of the cleavage reaction was carried out, we found that a significant proportion of cleavage products was detected within 90 min and continued to accumulate until 180 min (Fig. 3C). Thus, the overall reaction condition can be set at 30°C, pH 8.0, for 120 min.

Figure 3.

Figure 3.

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Optimization of the cleavage reaction and cleavage kinetics. The reaction conditions of temperature (A), pH (B), time (C), and ion concentrations (D and E) were tested as indicated. Nuclear extract prepared from wild-type Arabidopsis cell cultures were used for the temperature, time course, and Mg2+ optimization, while the remaining determinations were performed with extracts prepared from cultures overexpressing the AtCPSF73-I gene. F, The requirement of ATP for the cleavage reaction. The bands corresponding to cleaved products and uncleaved substrates were measured by ImageQuant (GE Healthcare Inc.). Cleavage ratio (CR) is calculated as the ratio between cleaved and uncleaved STS and is presented beneath the gels. G, The cleavage kinetics were calculated from a Lineweaver-Burk plot of the cleavage velocity determined at various template (substrate) concentrations. All the RNA substrates used in these assays were uniformly labeled.

It has been shown that divalent metal ions, such as Mg2+ and Mn2+, may affect cleavage and polyadenylation in both mammalian and yeast systems (Moore and Sharp, 1985; Wahle, 1991; Ryan et al., 2004). To test the efficacy of these ions in our cleavage system, uniformly labeled STS was incubated with nuclear extracts from AtCPSF73-I overexpressing cells under the optimal reaction conditions described above, except with varying concentrations of Mg2+or Mn2+ as indicated. The cleavage efficiencies were then compared. As shown in Figure 3D, cleavage efficiency increased with increasing Mg2+ concentration, until reaching the highest level at 6 mm. The cleavage efficiency remained high through the range of 3.5 to 6 mm Mg2+ (Fig. 3D). Thus, 3.5 mm was used as the standard condition. In contrast to Mg2+, replacing Mg2+ with Mn2+ (ranging from 0 to 20 mm) abolished cleavage activity (Fig. 3E), although it slightly promoted general RNA degradation from 0.32 to 2.5 mm. These results largely agree with those found in the in vitro assays of mammalian and yeast systems (Manley, 1983; Moore and Sharp, 1985; Butler et al., 1990), where Mg2+ is important for in vitro assays. ATP has an inhibitory effect on the cleavage activity starting from 1 mm, and total inhibition was reached at 2 mm, as shown in Figure 3F.

After optimizing the cleavage assay conditions, the reaction kinetics was studied. A series dilution of STS substrate was assayed using an excessive amount of the wild-type nuclear proteins under the standard cleavage assay conditions. The reaction was stopped after 30 min, the products were then resolved by a sequencing gel, and the ratio of cleaved products to uncleaved substrates were measured. Based on the amounts of RNA added, the concentrations of RNA substrate as well as the cleavage velocity were calculated. The Lineweaver-Burk plot shown in Figure 3G was drawn using the reverse of substrate concentration (1/[S]) versus the reverse of respective cleavage velocity (1/V). This exercise generated a Vmax of 8.4 nmol/min and Km of about 33.6 nm for STS pre-mRNA (202 nt).

Reconstitution of Cleavage Reaction Coupled with Polyadenylation by Supplementation with yPAP

Having successfully reconstituted cleavage activity, we extended our search for polyadenylation activity. However, after many attempts, no detectable poly(A) tail was found coupled with the cleavage activity. As in mammal and yeast mRNA 3′-end formation, the cleavage step is fully committed and irreversible; polyadenylation, on the other hand, requires additional protein factors, such as PAP and the poly(A) binding protein-II (Zhao et al., 1999). Since our extracts were made from the nucleus where polyadenylation occurs, it is likely that these proteins were there, but their activities were masked or inhibited. To circumvent this and reconstitute a fully functional 3′-end processing assay, we tested if yPAP could supplement the cleavage assay (Nu-CI) by adding poly(A) tails. To our surprise, the addition of yPAP extended the substrates for about 150-nts long in the presence of ATP (Fig. 4A, lane 2). When cordycepin (3′-dATP), an RNA chain elongation inhibitor, was added to the system at the same time yPAP was added, the extension of the newly cleaved product was stopped (lane 5, Fig. 4A), indicating that the observed extension is a product of yPAP [poly(A) tract]. To investigate whether the poly(A) addition was on the cleaved products, or the intact pre-mRNA, the polyadenylation products were cloned and sequenced after oligo(dT)-mediated reverse transcription-PCR. The results showed that, indeed, most of the poly(A) tails were added to the newly cleaved ends. Out of the 20 colonies sequenced, only four were from polyadenylation product of not cleaved substrate, while the remaining 16 were all from correctly cleaved substrates. These data indicated that 80% of the polyadenylated RNA were from substrates that were correctly cleaved.

Figure 4.

Figure 4.

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Reconstitution of cleavage reaction coupled with polyadenylation by supplementation with yPAP. A, Polyadenylation of cleaved STS by yPAP. Lane 1: Control reaction where buffer in place of Nu-CI or yPAP was added to template pre-mRNA; lane 2: incubation with both Nu-CI and yPAP for 2 h; lane 3: 2-h incubation with Nu-CI only; lane 4: 1-h incubation with yPAP after 2-h cleavage reaction by Nu-CI; lane 5: 1-h incubation with cordycepin (or 3′-dATP) and yPAP after 2-h cleavage reaction by Nu-CI; lane 6: 1-h incubation with yPAP without Nu-CI. B, A time course of cleavage and polyadenylation product formation when both Nu-CI and yPAP were added simultaneously to initiate the reaction. Pre-mRNA (uncleaved STS) and cleavage products are indicated by arrowheads. Polyadenylated RNAs are indicated by bracket. Incubation time (min) is noted on the top of each lane, and RNA size markers in nts are labeled on the right-hand side of each section.

PAP alone, however, could not add poly(A) to the pre-mRNA without the help of nuclear proteins (Fig. 4A, lane 6) under our assay conditions. This suggests that the specific addition of poly(A) is a result of the cooperation between the plant cleavage apparatus and yeast polyadenylation factors. It should be noted that similar results were also obtained by cleavage system using nuclear extract from wild-type cell culture (data not shown). The complementation further supports the idea that the cleavage activity described here is an authentic pre-mRNA cleavage since another polyadenylation factor, yPAP, recognizes and cooperates with it. In yeast, only the correct protein-protein interaction can lead to such functional cooperation (Meinke et al., 2008). Our study may indicate that a similar protein-protein cooperation is conserved in plant and yeast polyadenylation machinery. Such a cross-kingdom cooperative reconstitution of polyadenylation factors between yeast and plants opens up the possibility of complementation of other poly(A) factors, some of which were found to be impossible between mammal and yeast (Jenny et al., 1994, 1996).

The cooperative working model is further supported by the observation that the interaction of plant cleavage machinery with yPAP is required for efficient poly(A) tail addition. When yPAP was added after the cleavage step had already completed, poly(A) tails were shorter with uneven sizes, and many cleavage products remained unadenylated (compare Fig. 4A lanes 2 and 4). Such a progressive reaction by yPAP was also seen by a time-course study, where Nu-CI and yPAP were added at the same time and showed a uniform addition of poly(A) tails (Fig. 4B). It was also found that the reaction was progressive at 5 to 10 nt/10 min, but faster at the beginning at a rate of 60 nt/10 min (Fig. 4B).

DISCUSSION

We report here the successful establishment of an in vitro pre-mRNA cleavage assay system in Arabidopsis, in which yPAP can be add to the cleavage assay to achieve specific poly(A) tail addition. This assay system will not only promote the biochemical study of plant polyadenylation, but also shed light on the establishment of other mRNA processing in vitro reactions, e.g. splicing and transcription termination. Although genetic studies can explain how genes function in many cases, biochemical investigation certainly will lead to important functional information at the molecular level. With an increasing number of cases documenting gene expression regulation through alternative processing of pre-mRNA, such as alternative splicing and alternative polyadenylation, in vitro studies will be indispensable in understanding mechanisms at the biochemical level.

Compared to previous attempts to set up such an in vitro assay system, our success may be attributed to some distinct features such as the protein source, the method of preparing nuclear extracts, reaction conditions, and choice of pre-mRNA substrates. First, the nuclear proteins used in our study came from Arabidopsis suspension cells, which are actively dividing and growing cells, in contrast to materials used previously, such as young seedlings or cauliflower heads, both of which have only a small percentage of actively dividing/growing cells. The gene expression machinery in actively growing and dividing cells is continually active, and their nuclear proteins may therefore be more enriched with polyadenylation factors. Second, the method we used in our nuclear protein extraction may result in more abundant polyadenylation factors. Because the extraction began with a considerable amount of material (about 50-g cells), we can obtain about 0.5 to 1 g/L nuclear proteins, which is a relatively high concentration for in vitro assays, even after two centrifugations through Percoll gradients (to ensure purity). Meanwhile, we minimized the volume of nuclear extracts by directly adding ammonium sulfate to a final concentration of 0.5 m to break the nuclei. In the process, we also eliminated the need of using high-concentration ammonium sulfate to precipitate nuclear proteins, thus possibly help to preserve functional protein complexes that are required for the in vitro activity. Third, the assay conditions we used were carefully optimized to achieve maximum activity. In particular, the use of crowding agents such as polyvinyl alcohol and glycerol in the cleavage buffer may further increase the effective reaction concentration. Finally, the pre-mRNA substrate we used, CaMV STS pre-mRNA, is well characterized (Mogen et al., 1990; Rothnie et al., 1994). This 200-nt substrate contains both strong AAUAAA as a NUE, as well as the typical FUE, with an extension of a newly discovered CE (Loke et al., 2005) covering both sides of the cleavage site. These cis-elements may help cleavage site recognition and processing. Taken together, incremental modifications of all of these factors may contribute to a successful reconstitution of the cleavage assay.

Our in vitro cleavage assay system is quite similar to assays used for mammals and yeast, including assay conditions, a small volume reaction of about 10 μL, reaction over a period of 2 h at 30°C, and similar concentrations of Mg2+, K+, and Na+ ions. However, we did not include any EDTA in the cleavage buffer (not more than 0.10 mm carried over from the nuclear suspension buffer), and used much less radiolabeled RNA (only about 2,000 cpm or 2 pmol/reaction). Capping of the substrate RNA is not required for the cleavage activity (data not shown). While the poly(A) tail addition can be achieved by adding yPAP, the endogenous polyadenylation activity was not found in our assay. Possible reasons for this are that Arabidopsis PAP was not active or was inhibited by some factors, such as putative polyadenylation factor-B, which has been shown to inhibit nonspecific PAP activity (Forbes, 2004). It may be also due to the lack of correct interactions between PAP and other different polyadenylation factors, or the phosphorylation state of PAP (Bond et al., 2000). Meanwhile, other not-yet-identified PAP inhibitors may exist to prevent in vivo polyadenylation from happening. It is interesting to note that, initially, PAP activity in yeast in vitro polyadenylation assay system was very low when setting up; however, researchers ultimately determined how to achieve higher PAP activity (Butler and Platt, 1988; Butler et al., 1990).

With the supplement of yPAP, we managed to complement the polyadenylation activity in our assay system and provide further validation of our cleavage reaction at the same time. Some early work with polyadenylation factors indicated that such complementation with CPSF 73 and 100 and their counterparts in yeast (Jenny et al., 1994, 1996) was not successful. However, functional complementation of plant protein with yeast protein, or vice versa, is not uncommon (e.g. among exosome proteins [Chekanova et al., 2000]). It seems that whether a yeast polyadenylation factor can complement its plant counterpart is determined by how much these factors have remained structurally conserved during evolution. In the case of PAP, yeast and plants are relatively conserved with 40% identity in the first 400 amino acid sequences (Hunt et al., 2000). Our results provided direct evidence that at least some yeast polyadenylation-related proteins can work cooperatively in the plant system. It would be interesting to see if plant polyadenylation factors can complement the functions of other yeast counterparts.

Clearly, the establishment of such an in vitro cleavage assay system opens up a new avenue to study plant polyadenylation. Future directions should include the identification of the plant cleavage/polyadenylation factors that are responsible for the activities, and the roles of those potential factors revealed through bioinformatic and genetic studies, as confirmed by protein-protein interactions (Xu et al., 2006; Hunt et al., 2008; Xing et al., 2008a, 2008b; Zhao et al., 2009). Several approaches can be employed to identify the plant polyadenylation factors through this type of biochemical assay. Moreover, with the broad spectrum of tools we have generated, such as genetic mutants, gene expression, and protein interaction profiles, the assay system will undoubtedly promote the synergistic study of plant mRNA alternative polyadenylation, and posttranscriptional gene expression regulation in general.

MATERIALS AND METHODS

Arabidopsis Suspension Cell Cultures

Arabidopsis (Arabidopsis thaliana) suspension cell culture (MM1 from Landsberg erecta) was used in this research. Culture conditions, construction of AtCPSF73-I-TAP fusion proteins, and cultured-cell transformation were described elsewhere (Zhao et al., 2009).

Nuclear Protein Extract Preparation

Nucleus isolation from Arabidopsis suspension cultures was modified from Escobar et al. (2001) and Folta and Kaufman (2006). About 50-g suspension cells were ground in liquid nitrogen with acid-washed sand (Sigma-Aldrich Inc.) and then resuspended in the extraction buffer, and filtered through two layers of Miracloth twice. The filtrate was centrifuged at 25,000g for 5 min. The pellet was collected, resuspended in 30% Percoll (GE Healthcare), then overlaid on the top of 30% and 80% Percoll double layers, and centrifuged again at 2,000g for 30 min using a swing rotor. The middle layer between the 30% and 80% Percoll layers was collected, washed twice with buffer B (25 mm Tris-HCl pH 8.0; 10 mm MgCl2; 0.46 m Suc; 0.5 mm phenylmethylsulfonyl fluoride (PMSF); 6 mm β-mercaptal ethanol; 0.5% Triton X-100), and the nuclei were resuspended in 5 mL buffer C (25 mm Tris-HCl pH 8.0; 10 mm MgCl2; 0.46 m Suc; 0.5 mm PMSF; 6 mm β-mercaptal ethanol; 75% Percoll). The nuclei were then collected by centrifuge at 5,000g for 30 min. The concentrated nuclei were resuspended in buffer D (20 mm HEPES pH 8.0, 25% glycerol, 0.4 mm EDTA, 0.5 mm PMSF, 1 mm dithiothreitol, and 100 mm NaCl) and lysed by slowly adding a 2.0-m ammonium sulfate solution to a final concentration of 0.5 m. Lysed nuclei were centrifuged at 13,000 rpm for 30 min, and the supernatant (soluble nuclear protein extracts) was recovered and stored at −80°C freezer for future use.

Labeling of RNA Substrates for in Vitro Assays

The 3′-UTR of CaMV 35S RNA STS clone (Mogen et al., 1990) was a gift from Dr. Arthur Hunt (University of Kentucky). The target region (Fig. 1B) was amplified with a primer fused with a T7 promoter sequence at the 5′ end. The gel-purified PCR product was used as a template for in vitro transcription using the AmpliScrib T7 high yield transcription kit (Epicentre, Inc.), according to the manufacturer’s instructions. The same kit was used for the transcriptions of cold and [α-32P] ATP-labeled (using one-tenth of cold ATP) RNA. The cold STS RNA was 5′-end labeled by [γ-32P] ATP using RNA kinase (Epicentre, Inc.), and 3′-end labeled by [5′-32P] pCp using T4 RNA ligase (New England Biolabs, Inc.). All other templates (including At5g38420 and STS variants) were amplified by PCR, except the deletion mutants of FUE and NUE, which were produced by overlap extension PCR (Warrens et al., 1997). The labeled RNAs were purified using 7 m urea 6% polyacrylamide gel. The corresponding gel bands were cut out, eluted, precipitated, and stored for further use.

In Vitro Cleavage and Polyadenylation Assay

For an in vitro assay, in a 0.6-mL Eppendorf tube, 4.5 μL of cleavage buffer (5 mm MgCl2; 41.67 mm phosphocreatine disodium; 1.67 mm ATP; 3.3% glycerol; 0.8% polyvinyl alcohol; 3.3 mm HEPES pH 8.0), 0.5-μL RNaseOut (an RNase inhibitor; Invitrogen Inc.), and 0.5-μL nuclear protein extracts (about 0.5 μg/μL), diluted pre-mRNA substrate (1,000–2,000 cpm/reaction; typically about 2 pmol of RNA), and water were added to a total final volume of 7.5 μL. The reaction was incubated at 30°C for 2 h. For polyadenylation assays, 1 μL diluted (0.01×, about 6 units) yPAP (US Biochemical Inc.) was added. When the reaction was done, 7.5 μL 2× RNA gel-loading buffer was added, then all 15 μL was loaded to a 6% sequencing gel and run at 1,000 V for 1.5 to 2 h. The gel was transferred to Whatman filter paper, dried, autoradiographed, and scanned by a PhosphorImager scanner (Molecular Dynamics Inc.).

Sequencing of the Cleavage and Polyadenylation Products

Cleavage products of cold RNA were gel purified, and a 3′-end RNA linker (miRCat33; Integrated DNA Technology, Inc.) with a preactivated 5′ end and a blocked 3′ end was ligated to the 3′ end of the purified cleavage product, as described by the manufacturer. The ligation product was then reverse transcribed using a primer against the 3′-end linker. The resulting products were PCR amplified by primers; one matched the 3′-end linker, and the other matched the 5′ end of RNA. The amplified fragments were cloned into pTopo TA cloning vector (Invitrogen) and sequenced to reveal the cleavage sites. The polyadenylation product was reverse transcribed with oligo(dT)18VN primer with adaptor sequence, PCR amplified using an STS-specific primer and a primer annealed to the adaptor sequence, and then cloned using pTopo TA vector and sequenced.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AY140900.

Acknowledgments

We thank Arthur Hunt for the CaMV STS construct, other Li lab members for suggestions, David Martin and Tommy Li for language editorial assistance, and Miami University Instrumentation Laboratory and the Center for Bioinformatics and Functional Genomics for technical support.

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