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Characterization of a novel plant poly(A) polymerase
Plant Science 110 (1995) 215-226
ELSEVIER
Characterization
of a novel plant poly(A) polymerase
Jaydip Das Gupta, Qingshun Li, A. Brian Thomson, Arthur G. Hunt* Plant Physiology/Biochemistry/Molecular Biology Program, Department of Agronomy, Universiry of Kentucky. Lexington, KY 40564-0091. USA
Received 13 March 1995; revision received 8 June 1995; accepted 10 July 1995
We have purified and characterized poly(A) polymerases (PAPS) from Pisum sativum, Brassica juncea, and Zea mays. Through chromatography on DEAE-Sepharose and heparin-Sepharose, these PAPS copuritied as a single enzyme along with RNPs that could provide RNA substrates for the enzyme. More extensive purification by chromatography on MonoQ resulted in the resolution of the PAPS into as many as three fractions. One of these (PAP-l) contained a 43-kDa polypeptide immunologically related to the yeast PAP, and two others (PAP-II and PAP-III) contained RNAs that could serve as substrates for polyadenylation. These fractions by themselves possessed little PAP activity, but mix-
tures containing combinations of these displayed substantial activity. Similar PAP factors (PAP-l and PAP-III) were identified after fractionation of extracts prepared from Brassica juncea and Zea mays. The factors from one plant were completely interchangeable with those from different plants. We conclude that the poly(A) polymerases present in vegetative plant tissues consist of more than one component. In this respect, they are substantially different from other reported plant, mammalian, and yeast PAPS. Keywords: mRNA Polyadenylation;
RNA processing; Posttranscriptional
1. Introdnctioo
Messenger RNA 3 ’ end formation in eucaryotes is an RNA processing event involving an endonucleolytic cleavage at the so-called polyadenylation site, followed by the addition of a polyadenylate tract to the 3 ’ end of the processed RNA [ 11. In mammals, pre-mRNAs are processed by a set of factors that recognize a polyadenylation
* Corresponding author, Tel.: (+1-606) 257 3637; Fax: (+l606) 323 1952.
signal (AAUAAA) located upstream from the cleavage/polyadenylation site, and a UG-rich element located downstream from this site. These factors mediate the site-specific cleavage of the pre-mRNA and subsequent polyadenylation of the cleaved RNA. The polyadenylation reaction is catalyzed by a PAP and proceeds in two stages: the first entails the addition of about 10 nts, and requires the polyadenylation signal AAUAAA in the substrate RNA molecule; the second results in the addition of some 200 nts and involves, in addition to the PAP, a nuclear polyadenylate binding protein [2].
0168-9452/95/$0X50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(95)04205-Z
control
Characteristic PAPS are responsible for the addition of the polyadenylate tracts to the 3’ ends of mRNAs in mammalian and yeast cells The purified calf thymus enzyme consists of a mixture of polypeptides ranging between 57 and 60 kDa [3]; this protein is the product of a gene capable of encoding polypeptides of 77 [4] or 82 kDa [5], and the size of the protein purified from calf thymus has been attributed to proteolysis during purification [4]. This enzyme can polyadenylate RNAs non-specifically (e.g. independent of a polyadenylation signal) in the presence of Mn’+ [3] and acts in concert with other factors to process and polyadenylate precursor mRNAs in a poly(A) signal-dependent manner [l]. The purified yeast enzyme is a 63-kDa polypeptide that can also polyadenylate RNAs non-specifically [6]; with this enzyme, Mn*+ and Mg*+ can satisfy the divalent metal cation requirement. The yeast enzyme is one of several factors required for poly(A) signaldependent polyadenylation of precursor mRNAs
[71. Plant polyadenylation signals differ substantially from mammalian signals. In plant genes, a complex series of cis elements in a pre-mRNA is needed for efficient mRNA 3 ’ end formation [B]; these include elements that may be functionally analogous to the mammalian polyadenylation signal AAUAAA (termed near-upstream elements, or NUEs, in plant genes), the cleavage/polyadenylation site (CS) itself, and one or more novel elements (far-upstream elements, or FUEs) located upstream from the putative AAUAAA-like motifs. To better understand the process of mRNA 3 ’ end formation in plants, we have initiated biochemical studies focusing on possible polyadenylationrelated activities. Here, we describe the characterization of PAPS from three different plants. These enzymes can be chromatographically resolved into several fractions. One of these contains a polypeptide that is immunologically related to the yeast PAP; however, this fraction is not capable of polyadenylating exogenously added RNA. The other fractions contain populations of RNPs whose RNAs can serve as substrates for the PAP. Our studies indicate that the plant PAPS can be resolved into more than one component, and are thus different from PAPS isolated from other organisms.
2. Methods 2.1. Plant materials Garden pea (Pisum sativum cv. Laxton Progress) seed was obtained from Kentucky Garden Supply (Lexington KY). Seeds were sown in the greenhouse and harvested after 2-3 weeks of growth, when plants had two to three extended open trifoliates. Corn (Zea mays, Pioneer 524) was obtained from Dr Michael Barrett (Department of Agronomy, University of Kentucky); seeds were sown in the greenhouse and harvested after 8 days of growth. Indian mustard (Brassica juncea) was obtained from Dr R.K. Mandal (Department of Biochemistry, Bose Institute, Calcutta, India); seeds were sown in the greenhouse and harvested after 10 days. 2.2. RNA substrates Poly(A), average length - 400 nts, was obtained from ICN Biochemicals. To produce RNAs containing the wild-type rbcS-E9 polyadenylation signal, nts +703 through +1002 of the pea rbcS-E9 gene [9] were cloned as an NsiI-HaeIII fragment into PstI+EcoRV-digested Bluescript. Transcription using this plasmid (rbcS-E9-wt) as template yielded RNAs containing the complete rbcS-E9 polyadenylation signal [lo]; these RNAs are denoted as rbcS-EPwt RNAs in this paper. One additional RNA (termed ‘precleaved’, because it contains all of the sequences that should be present in a pre-mRNA that has been cleaved at a polyadenylation site but has not yet been polyadenylated) was prepared directly from a PCR-amplified template. Fifty nanograms each of two primers (5 ‘-TAATACGACTCACTATAGGGGTTTCGACAACGTTCGTCA-3’ and 5’TGAGAATGAACAAAAGGACC-3 ‘), approximately 50 ng of a plasmid containing the wildtype rbcS-E9 polyadenylation signal (pGB 1B) [B], 5 ~1 of 10 x Taq DNA polymerase buffer (1 x = 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgC12 and 0.01% gelatin) were brought to a volume of 50 ~1 with Hz0 and Taq DNA polymerase (0.2 units) was added. DNAs corresponding to nts -215 to +l of the rbcSE9 polyadenylation signal were amplified (35 cycles, each consisting of 1 min at 92°C 1 min at 45°C. and 2 min at 72”(Z), the PCR products were ex-
J. Das Gupta et al. /Plant Science II0 (199.0 215-226
tended at 72°C for 5 min and then purified by electrophoresis in agarose gels and elution from ion exchange paper. Approximately 1 fig of PCR product was used in each transcription reaction, as described above. Each RNA was used as described in the text for PAP reactions. Additionally, these were tested for ability to serve as substrates for the yeast PAP; in all cases examined, the in vitro transcripts proved to be suitable RNA primers for polyadenylation using the yeast enzyme (data not shown). 2.3. Poly(A) polymerase reactions The non-specific PAP assay was a modification of that described by Rose and Jacob [l I]. Unless otherwise indicated, the fraction of interest (5-20 ~1)was brought to a total volume of 50 ~1with buffer I (40 mM KCl, 25 mM Hepes-KOH pH 7.9, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10% glycerol and 5 pg/ml of each of leupeptin, chymostatin, and antipain). This was mixed with 9 ~1of PAP reaction mix (167 mM Tris-HCl pH 8.0, 267 mM KCl, 3.33 mM MgCI,, 0.33’mM EDTA, 3.33 mM DTT, 0.67% nonidet-P40, 1.28 mg/ml bovine serum albumin, 3.33 mM ATP, and l-2 rCi of [CY-3ZP]ATP). When indicated, poly(A) was added to a final concentration of 3.33 mg/ml and unlabeled &S-derived RNAs to a final concentration of 2.5 PM. Reactions were carried out at 30°C for 2 h and then stopped by extraction with phenol/chloroform/isoamyl alcohol (25:24: 1, v/v/v). Of the aqueous phase, 20 ~1 were spotted on a l-cm* piece of DE-81 filter paper (Whatman), the filters washed five times in 5% Na2HP04, and the incorporated radioactivity was measured by liquid scintillation spectrometry. In some instances, the products of polyadenylation reactions were analyzed on sequencing gels. The aqueous phases of the terminated reactions were precipitated with ethanol and the nucleic acids dried and analyzed on 6% sequencing gels. Gels were dried and visualized by autoradiography using Kodak XAR-5 film. 2.4. Analysis of endogenous RNAs Endogenous RNAs present in the various PAP preparations were isolated by extraction with phenol/chloroform/isoamyl alcohol and precipitation with ethanol. These RNAs were end-labeled
217
with purified yeast PAP (US Biochemicals) using the protocol suggested by the manufacturer. Briefly, 1 pg of RNA was incubated in a 30-~1 reaction containing 6 ~1 of the supplier’s 5 x reaction buffer (100 mM Tris-HCI pH 7.0, 250 mM KCI, 3.5 mM MnC12, 1 mM EDTA, 500 &ml acetylated bovine serum albumin, and 50% glycerol), l-2 &i of [ar-32P]ATP (1500-3000 Ci/mmol), and 100 units of yeast PAP. After 30 min at 30°C the reactions were terminated by extraction with phenol/chloroformIisoamyl alcohol, and nucleic acids recovered by alcohol precipitation. These were then separated on 6% sequencing gels. Gels were dried and visualized by autoradiography using Kodak XAR-5 film. Under these conditions, very short (20 nts or less) oligoadenylate tracts are typically added by the yeast PAP [6]. 2.5. RNAse A digestions and alkali treatment of nucleic acids To treat the products of polyadenylation by the pea and yeast enzymes with RNAse A, the RNAs recovered from a single reaction (typically, between lo4 and lo5 counts/min) were dissolved in 50 ~1 of 10 mM Tris-HCl pH 8.0 + 1 mM EDTA (TE), 5 ~1 of RNAse A (40 units/pi, from US Biochemicals) were added, and reactions carried out at 37°C for 30 min. Reactions were terminated by extraction with phenol/chloroform/isoamyl alcohol, tRNA was added to a concentration 100 &ml, and the nucleic acids recovered by alcohol precipitation and analyzed on sequencing gels as described above. To treat the products of the pea PAP reactions with alkali, the RNAs recovered from a single reaction were dissolved in 20 ~1 of TE as described in the preceding paragraph. Five microliters of this were mixed with 20 ~1 of TE and 25 ~1 of 2 N NaOH. After 30 min at room temperature, this solution was extracted with phenol/chloroform/ isoamyl alcohol (saturated with Tris-HCl pH 7.9), tRNA was added to a concentration of 100 pg/ml, and the nucleic acids recovered by alcohol precipitation and analyzed on sequencing gels as described above. 2.6. Purification of plant PAPS PAPS were isolated from green leaves of young plants. Unless otherwise indicated, all operations
218
J. Das Gupta CI (11./ Plunt Science
were done at 4°C. Leaves were harvested, chopped to tine pieces, and placed in grinding buffer (110 mM KCl, 15 mM Hepes-KOH pH 7.9, 5 mM MgCl*, 0.1 mM EDTA, I mM DTT, 1 mM PMSF, 9.4% w/v ammonium sulfate and 5 pglrnl of each of the protease inhibitors leupeptin, chymostatin, and antipain; 2 ml of buffer was used for each gram of leaf material). Leaves were then homogenized for 30 s using a Polytron tissue homogenizer (with 0.1% octanol as an antifoaming agent), the homogenate filtered through four layers of cheese-cloth, and the filtrate centrifuged at 7000 x g for 30 min. Solid ammonium sulfate was added to bring the supernatant to 35%. The solution was stirred for 30 min and centrifuged at 2000 x g for 10 min. Solid ammonium sulfate was then added to bring the supernatant to 70% saturation and the solution was stirred for 30 min and centrifuged as before. The resulting pellet was resuspended in buffer I. The extract was then dialyzed for 12- 16 h against a buffer (dialysis buffer) containing 40 mM KCl, 25 mM Hepes-KOH pH 7.9, 0.1 mM EDTA, 1 mM PMSF, 10% glycerol, and 12 mM fi-mercaptoethanol. After dialysis, the extract was stored at -80°C. A total of 500 mg of protein was loaded onto a 70-ml DEAE-Sepharose column (Sigma) that had been equilibrated with buffer I. The column was washed at 1 ml/min with 100 ml of buffer II (buffer I containing 200 mM instead of 40 mM KCI) and proteins that were retained on the matrix eluted with a IOO-ml gradient (200-500 mM) of KCI in buffer I. Fractions of 5 ml were collected and assayed for PAP activity without dialysis. Fractions containing active PAP were pooled and dialyzed against dialysis buffer. A total of 25 mg of protein was then loaded onto a 15-ml heparinSepharose (Pharmacia) column that had been equilibrated with buffer I. The column was washed with 35 ml of buffer I and 5-ml fractions collected. Proteins that were retained on the matrix were eluted with 50 ml of buffer III (buffer I containing 500 mM instead of 40 mM KC]). Fractions of 6 ml were collected and assayed for PAP activity without dialysis. PAP-containing fractions were stored at -80°C. The heparin-Sepharose flowthrough, which had a majority of the PAP activity, was loaded at 0.25
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ml/min onto a Mono-Q HR5/5 column (Pharmacia) that had been equilibrated with buffer I. This column was developed with a 20-ml linear gradient of KC1 (40-500 mM) in buffer I and l-ml fractions collected. Fractions were assayed for activity without dialyzing, and active fractions corresponding to PAP-I, -11, and -III (see Section 3, Results) were pooled and dialyzed against dialysis buffer. At this stage, the different PAP fractions had protein concentrations between 50 and 250 pg/ml. The PAP fractions were divided into 100~~1 aliquots and stored at -80°C. For further purification, 500 ~1 (approximately 10 pg) of Mono-Q purified PAP-I were separated, without prior dialysis, on Superose 6 columns. The sample was loaded, and the column developed, with buffer I at a flow rate of 0.25 ml/min. Onemilliliter fractions were collected and assayed for activity or analyzed by SDS-PAGE. 2.7. Micrococcal nuclease treatment of PAP-III A total of 100 ~1of PAP-III (after MonoQ chromatography) was brought to 5.6 mM in CaCIz by adding 11.2 ~1 of a 50 mM stock solution. Micrococcal nuclease (6 ~1 of a 15 I-J/~1stock solution) was added and the mixture incubated at 30°C for 30 min. A total of 14 ~1 of 50 mM EGTA (pH 7.0) was added to stop the micrococcal nuclease digestion and 20 ~1 of this mixture used directly in the PAP assay. For this treatment, controls in which the micrococcal nuclease was replaced with water were done as well. Treatments of labeled RNAs in the buffers used here indicated that micrococcal nuclease was active in these conditions and was capable of hydrolyzing >90% of input labeled RNAs. In some cases, micrococcal nuclease-treated PAP-III was extracted with phenol/chloroform/ isoamyl alcohol and the residual nucleic acids precipitated with ethanol and characterized as described above. 2.8. hmunoblot analysis For Western blots, proteins were separated by SDS-PAGE and the separated proteins transferred to a nitrocellulose membrane [ 121 using a TransBlot Cell (Bio-Rad Laboratories) following the manufacturer’s recommendations. Gels were
J. Das Gupta et al, /Plant
either stained with Coomassie Brilliant Blue [13] or probed with monoclonal antibodies that recognize an epitope in the C-terminal portion of the yeast PAP [14]. For the latter, filters containing transferred proteins were incubated (at room temperature) in TTBS for 5 min, TTBS containing 5% (w/v) nonfat dried milk for 1 h, and then with 5% milk-TTBS containing a 1:5000 dilution of monoclonal antibody for 12-16 h. The monoclonal antibody was removed with three washes (5 mm each) of TTBS, the filters incubated for 3 h with 5% milk-TTBS containing goat anti-mouse IgG alkaline phosphatase conjugate (Sigma), and this antibody conjugate removed with three washes of TTBS. Filters were equilibrated in alkaline phosphatase buffer (0.1 M Tris-HCl pH 9.5, 0.1 M NaCl, 0.05 M MgCl,) and developed in alkaline phosphatase buffer + 2 nitroblue mg/ml tetrazolium + 1.2 mg/ml 5-bromo4chloro-3indoyl phosphate; development was stopped by washing with deionized water.
Science I10 (1995) 215-226
219
crude extract 0.2 M
+-I
DEAE-Sepharose
ca. 0.35 M DEAE-PAP 0.5 M
heparin-Sepharose 0.04 M
I+ HS-PAP
MOIIOQ 0.25 M
0.36 M
0.43 M
4 PAP-I
4 PAP-II
4 PAP-III
Fig. 1. Illustration of the purification strategy for the pea PAP.
1.2 flfl
3. Results 1.0
3.1. Purification and characterization of a pea PAP It is difficult to measure PAP activity in crude pea extracts by following the modification of labeled input RNAs, presumably due to the presence of large quantities of non-specific nuclease activity in these extracts (B.D. Mogen and A.G. Hunt, unpublished observations). We therefore set out to characterize PAPS in these extracts using a nonspecific polymerization assay [l I]. This assay measures the incorporation of [or-32P]ATP into products that can be immobilized on ion exchange paper and is well suited measuring PAP activity in the presence of nucleases. Using this assay, potential PAP activity could be detected in crude extracts prepared from pea seedlings. Consequently, fractions containing possible PAP activity were obtained by chromatography on DEAESepharose and heparin8epharose (summarized in Fig. 1). The active fractions after heparin8epharose chromatography were by all accounts a poly(A) polymerase. In particular, this activity was inhibited by 3’-dATP (Fig. 2), presumably as a result of chain termination. Moreover, polymeriz-
0.2 0.0 pdy(A)
+
Mg2+ Mn2+ 3’-dATP
+
+
-
-
-
+
-
+
I
+
-
+
-
+
-
+
I
-
-
+
-
-
+
Fig. 2. Properties of the HS-PAP. Reactions were performed as described in Section 2, Methods, with the exceptions noted beneath the graph: poly(A) was added to some reactions to a final concentration of 3.33 mg/ml, MgCI, was replaced with MnCl* (0.5 mM), or ATP was replaced with a mixture of unlabeled 3’-dATP (0.5 mM) + 1-2 &i of [~Y-~~P]ATPper reaction. Reactions contained IO ~1 (5.4 fig) of HS-PAP and were incubated at 30°C for 2 h before extraction and quantitation.
J. Das Cupta et al. /Plant Science I10 (1995) 215-226
220
ing activity was not apparent when [CX-“PI3‘dATP, [w~~P]CTP, [a- 32P]GTP, or [w~~P]UTP were used as nucleotide substrates (not shown). This activity, termed the HS-PAP, displayed a marked preference for Mg2+ over Mn2+ (Fig. 2) and had an apparent KM for ATP of 39 f 1.5 PM in the presence of 0.5 mM Mg2+. However, in these studies, addition of poly(A) (Fig. 2) or rbcSEPderived RNAs (data not shown) had little effect on PAP activity; indeed, the HS-PAP was remarkably active in the absence of added RNA (Fig. 2). The activity in the absence of exogenously added RNA suggests the presence of endogenous RNA in the HS-PAP. However, other possible reactions could account for the production of labeled products able to bind to ion exchange paper. Thus, to assure that the labeled products we measured were the products of a polyadenylation reaction, the products obtained from reactions
lacking exogenous added RNA were characterized. After extraction with phenol and chloroform, the labeled products were analyzed on sequencing gels (Fig. 3). A population of large nucleic acids was evident after autoradiography; these nucleic acids were susceptible to RNAse A (Fig. 3A) and alkali (Fig. 3B), indicating that they were RNA. Moreover, after RNAse A digestion, these products had a broad size distribution (Fig. 3A). Since RNAse A does not digest polyadenylate tracts, this result indicates the presence of extended poly(A) tails in the molecules labeled by the HSPAP. These data rule out the presence of any activities that might initiate poly(A) formation de novo (as these would not be affected by treatment with RNAse A) and suggest that the HS-PAP contains an endogenous population of RNAs that can serve as substrates for this enzyme. Further attempts to purify the pea PAP consistently resulted in a dramatic loss of activity.
B.
A. RN Ase
Ar:
-
+
alkali:
-
+
364
242
Fig. 3. Sequencing gel analysis of RNAs produced by HS-PAP. Polyadenylation reactions were performed with 10 ~1 (5.4 pg) of HSPAP, [w~*P]ATP and no added RNA and were incubated at 30°C for 2 h. The labeled products isolated by phenol/chloroform extraction and ethanol precipitation. (A) One sample was analyzed on a sequencing gel before and one after RNAse A digestion. Positionsof DNA size standards are shown on the left. (B) One sample was analyzed on a sequencing gel before and one after treatment with alkali. Positions of DNA size standards are shown on the left.
J. Das Gupta et al. /Plant Science I10 (199.5)215-226
After MonoQ chromatography, less than 5% of the activity loaded onto the column was recovered. One possible explanation for this loss of activity was that the HS-PAP was actually a multicomponent enzyme, the individual components of which were resolved by chromatography on MonoQ. To test this, we systematically assayed each MonoQ fraction for activities that stimulated the PAP recovered from this column. In this manner, three components, designated as PAP-I, PAP-II, and PAP-III in respective order of their elution from the MonoQ column, could be identified (Fig. 1). Interestingly, the activity of the reconstituted enzyme at this stage of purification was still largely independent of exogenously-added RNA, indicating the presence of endogenous RNAs in one or more components. In some preparations, all three components proved to be required for optimal activity (Table 1, Experiment 1). In others, however, maximal activity could be observed with just the combination of PAP-I + PAP-III (Table 1, Experiment 2). For this reason, further experiments focused on PAP-I and PAP-III. 3.2. PAP-I contains a polypeptide that is immunologically related to the yeast PAP
The above studies indicate that the PAP activity in pea extracts is capable of utilizing RNAs present in endogenous RNPs, but they do not identify which of the three PAP components includes the
221
catalytic moiety responsible for actual polyadenylation. In an attempt to determine this, each component was analyzed by immunoblotting using a monoclonal antibody raised against the yeast PAP. This experiment revealed the presence, in PAP-I, of a 43-kDa polypeptide that was recognized by the monoclonal antibody (Fig. 4B). Equally abundant polypeptides in PAP-II and -111 (Fig. 4A) were not recognized (Fig. 4B), and omission of the monoclonal antibody from the procedure eliminated the appearance of the band seen in Fig. 4B (data not shown). These results indicate a specific interaction between the 43-kDa polypeptide and the antibody raised against the yeast PAP. This 43-kDa polypeptide was consistently seen in several (> 10) different Mono-Q PAP-I preparations, and was one of two polypeptides that coactivity on chromatographed with PAP-I Superose-6 columns (Fig. 5). Taken together, the results presented in Figs. 4 and 5 suggest that the 43-kDa polypeptide seen in PAP-I is analogous to the yeast PAP. However, PAP-I was unable to polyadenylate exogenously added RNAs (Table 2), including RNAs that contain an intact polyadenylation signal (rbcS-E9-wt) and RNAs that resemble the putative products of pre-mRNA cleavage by the polyadenylation ap-
Table I Reconstitution using PAP-I, II and III Enzyme
Experiment I
Experiment 2
HS PAP PAP-I PAP-II PAP-III PAP-1 + II PAP-I + III PAP-II + III PAP-I + II + III
35 894 176 1077 1224 2529 20 378 19094 63411
ND 0 121 504 429 12 137 4428 9580
Enzyme reactions were performed as described in Section 2, Methods, with IO ~1 (5.4 rg) of HS-PAP or with 5 ~1 of PAP-I, PAP-II and PAP-III, as indicated. The results of two experiments, done with different preparations of PAP, are shown as counts/mm retained on DE-81 paper, corrected for background. ND, not determined.
Fig. 4. SDS-PAGE and immunoblot analysis of PAP-I, -II and -III. Aliquots of each PAP component were separated on duplicate 12% acrylamide gels containing SDS. One gel (A) was stained with Coomassie Brilliant Blue [I31 and the other (B) probed with a monoclonal antibody specific for the yeast PAP [l4]. The immunoreactive band in part B corresponds in size to the principal stainable band in PAP-1 in part A.
222
J. Das Gupta et al. /Plant
PAP activity fraction
5.
21
20
19
18
Science 110 (19%)
17
16
.?l.f-226
15
g
M 218
102 72 43 29 18
Fig. 5. Superose 6 purification of PAP-I. PAP-I obtained after Mono-Q chromatography was further purified by chromatography on Superose 6. Aliquots (40 ~1) of each fraction were assayed for PAP-I activity in the presence of PAP-III (10 al). Alternatively, aliquots (100 cl) were separated on polyacrylamide gels containing SDS. which were subsequently stained with Coomassie Brilliant Blue 1131.The polypeptide that is recognized by monoclonal antibodies raised against the yeast
Table 2 PAP-I cannot polyadenylate exogenously added RNAs RNA
Counts/min
None Poly(A) rbcS-E9-wt rbcS-E9-PC PAP-III
0 7 0 0 13 28l=
Reactions were performed as described in Section 2. Methods, with IO ~1 of PAP-I. PAP-III (IO ~1) or the indicated RNAs were added to the respective reactions and PAP activity determined as described in Section 2, Materials and methods. Results are given as counts/mm retained on DE-81 paper, corrected for background. ‘In the absence of PAP-I, PAP-III incorporated a net of 186 counts/mm into poly(A).
PAP (Fig. 48) is indicated by the arrow on the left and the sizes of molecular weight standards on the right. The column fractions are denoted above their corresponding lanes, as are lanes containing Mono Q-purified PAP-I (onto) and molecular weight size standards (M). The PAP activity measured after mixing each fraction with PAP-III is depicted above the lane designations; for reference, the activities in fractions 17, 18, and 19 were 33 165, 46 477 and 7660 counts/min, respectively.
paratus (rbcS-E9-PC). Thus, although PAP-I contains a polypeptide that is immunologically related to the yeast PAP, it is not itself the sole component of the corresponding plant enzyme. Instead, additional factors present in PAP-III are required for activity.
3.3. PAP-III contains endogenous RNAs As was the case with the HS-PAP, the reconsitution of PAP activity with MonoQ-purified components did not require added RNA (Table 1). This observation suggested the presence of RNA in one or more of these components. Accordingly, aliquots of PAP-I, -11, and -111were extracted with phenol/chloroform extraction and possible nucleic acids recovered by ethanol precipitation. About 50 ng of nucleic acid per c(g of protein could be
J. Das Gupta et al. /Plant
recovered from MonoQ-purified PAP-II, and 250 ng of nucleic acid per pg of protein from PAP-III. In contrast, no nucleic acid (< 10 rig/g of protein) could be recovered from PAP-I. The nucleic acids recovered from PAP-III had a broad size distribution (Fig. 6, lane 1). In some preparations, as shown in Fig. 6, these nucleic acids were relatively small, ranging between 20 and 150 nts. In other preparations, nucleic acids as large as 400 nts could be observed (not shown). These observations indicate that the nucleic acids present in PAP-III are heterogeneous in terms of size. These nucleic acids were susceptible to digestion with RNAse A (Fig. 6, lanes 2 and 4). Therefore, PAPIII contains a population of RNAs with a broad range of sizes. This result confirms the suggestion regarding endogenous RNAs that was inferred from the experiment shown in Fig. 3. Similar results were obtained with preparations of PAP-II (data not shown). The RNAs present in PAP-II and PAP-III survive an extraction and purification process that exposes them to considerable endogenous nuclease activity. This suggests an association with one or more proteins in a ribonucleoprotein (RNP) complex. To examine this possibility, PAP-III was treated with micrococcal nuclease, and the treated samples examined for nucleic acid content and for PAP activity in the presence of PAP-I and absence of added RNA. Treatment of PAP-III with micrococcal nuclease had no discernible effect on the size distribution of RNAs present in PAP-III (Fig. 6, compare lanes 1 and 3). Moreover, the combination of PAP-I and micrococcal nucleasetreated PAP-III retained 77% of the activity of the combination of PAP-I and untreated PAP-III (not shown). Therefore, the endogenous RNAs in PAPIII were largely resistant to micrococcal nuclease. This result indicates the presence of a population of RNPs in PAP-III, the RNAs of which can serve as substrates for the reconstituted PAP. 3.4. Other plants have PAP-I and PAP-III that can substitute far the pea PAP-I and PAP-III me fractionation of the pea PAP into at least two components is an unusual characteristic not seen with PAPS from other organisms. Moreover, pips from other plants have been described, en-
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1234 147 118
67
27
Fig. 6. Characterization of nucleic acids present in PAP-III. RNA was isolated from PAP-III before (lanes I and 2) or after (lanes 3 and 4) treatment with micrococcal nuclease and endlabeled with purified yeast PAP. Shown are the products of labeling using the yeast PAP and [3ZP]ATP, before (lanes I and 3) and after (lanes 2 and 4) RNAse A digestion. Positions of DNA size standards are shown on the left. The labeled molecules remaining after RNAse A digestion are less than IO nts in size, and their low abundance indicates that the majority of label seen in the lane without RNAse A digestion was incorporated as very short oligoadenylate tracts, tracts that were not recovered by ethanol precipitation following RNAse A treatment.
zymes that can be purified as single polypeptides [15-181. It was thus possible that the PAP we purified was one unique to peas, for any of a number of reasons. To examine this possibility, we fractionated extracts prepared from Brassica juncea and Zea mays. Chromatographic profiles of PAP activity in these extracts mirrored those seen with pea extracts: PAP activity bound to DEAE columns and was present in the flow-through of
J. Das Gupta el al. /Plan! Science 110 (1995) 215-226
224
125
El corn PAP-III El mustard PAP-III pea PAP-III 25
none
corn
mustard
pea
PAP-I Fig. 7. Interchangeability of PAP-I and PAP-III isolated from different plants. PAP-I and PAP-III was isolated from maize, mustard and pea as described in the text. Aliquots of IO pl were tested for activity in the combinations shown. The activity of each PAP-III preparation in the absence of PAP-I (none) was compared with the activities measured after combining with the maize, mustard and pea PAP-I. Reactions were performed as described in Section 2, Methods (note that no exogenous RNA was added in these reactions). The activity of each PAP-I preparation in the absence of PAP-III was indistinguishable from background. The data shown were corrected for background (- 350 counttimin)
columns (data not shown). Moreover, these preparations utilized endogenous RNAs as substrates, much as did the pea enzyme. Further fractionation of the PAPS from these other plants by chromatography on MonoQ also yielded results similar to those seen with the pea enzyme. For each enzyme preparation, components with chromatographic properties similar to PAP-I and PAP-III could be seen. These separated components by themselves had little PAP activity (with or without exogenously added RNA), but when combined yielded substantial activity (Fig. 7). Moreover, the respective components were completely interchangeable: PAP-I from all plants was able to reconsitute PAP activity with the PAP-III from peas, etc. (Fig. 7). Therefore, the three plants examined all possess a functionally equivalent PAP, one that can be chromatographically resolved into more than one component.
heparin-Sepharose
4. Discussion We have characterized a PAP from several plants that is distinctive in many respects. It can be resolved after chromatography into at least two components. One of these (PAP-I) has no inherent PAP activity but consists of a polypeptide that is immunolgically related to the yeast PAP (Fig. 4) and copurifies with PAP-I activity (Fig. 5). Another component (PAP-III) has a low inherent activity that is markedly stimulated by PAP-I (Table 1). This component also includes a population of RNPs (Fig. 6), the RNAs of which can serve as substrates for the reconstituted PAP (Figs. 2, 3). The fact that different plant PAPS can be resolved into more than one component, each of which is required for activity, distinguishes these enzymes from other reported PAPS [3,6,15- 181. It is possible that this result is a consequence of protease action during the purification process, action that
J. Das Gupta et al. /Plant Science I IO (1995) 215-226
cleaves a larger PAP into two smaller polypeptides. The observation that PAP-I and PAP-III from different plants are functionally interchangeable (Fig. 7) argues against this possibility, as this would require the presence of a relatively specific, conserved proteinase in the three plant species studied here. However, we cannot completely rule out the action of a proteinase as an explanation of the multi-component nature of the PAPS we have studied. PAP-III contains a significant quantity of RNA (Fig. 6) that can serve as substrates for polyadenylation (Figs. 2, 3; Tables I, 2). These RNAs survive exposure to endogenous nucleases and treatment of PAP-III with micrococcal nuclease (Fig. 6, lane 3), indicative of their residing in an RNP. This feature of PAP-III raises two interesting possibilities regarding the nature of plant PAPS. It is possible that PAP-III includes, along with these RNPs, an additional polyadenylation factor that copurities with the RNPs (perhaps one that is derived from the action of proteases, as suggested above). Thus, the combination of this factor and PAP-I would be required for PAP activity, and the RNAs present in PAP-III would serve as convenient, available substrates for the reconstituted enzyme. Alternatively, it is possible these plant PAPS consist of an enzyme (PAP-I) and an associated group of RNPs (PAP-III) that serve as the preferred or sole RNA substrates for PAP-I. Further purification and the cloning of genes encoding different polypeptide components of PAP-I and PAP-III will be needed to completely distinguish between these possibilities. In most purifications, an additional component (PAP-II) can be identified (Table 1). This component has readily detectable inherent activity and contains substantial RNA. We presume that PAPII is a combination of PAP-I and PAP-III, and its chromatographic behavior on MonoQ is consistent with this presumption. However, we cannot rule out other explanations for the properties of this component, and further study will be needed to better understand this component. PAPS from a number of different plants have been described by others. Tarui and Minamikawa purified a PAP from germinated cowpea seeds [15,16]. This enzyme consisted of a 63-kDa poly-
225
peptide [IS] that had both PAP and poly(A)hydrolyzing activity [16]. PAPS from mung bean [ 171and wheat [ 181have been identified and found to consist of single polypeptides with molecular weights ranging of 30 kDa (for the mung bean enzyme) or 70 kDa (for the wheat enzyme). All the plant enzymes identified to date displayed a complete dependence upon exogenously added RNA and a marked preference for Mn2+ over Mg2+. The enzymes we describe here have no detectable poly(A) hydrolyzing activity (unpublished results), prefer Mg2+ over Mn2+, and copurify with a population of endogenous RNPs that can serve as the RNA substrate for PAP activity. It is thus likely that the enzymes we have characterized are different from the PAPS from cowpea, mung bean, and wheat germ. In the latter two respects, the enzymes we have characterized also differ from the purified mammalian and yeast enzymes [3,6]. The reasons for the differences between the PAPS we describe here and plant PAPS described by others are not known. Different PAPS may be present at different developmental stages in the growth of plants. The PAPS described here were isolated from leaves of plants that had been grown for l-3 weeks, whereas the PAPS identified in other plants were purified from embryonic tissues. The differences between the PAPS we describe and other plant PAPS may thus be due to the involvement of different PAPS in early development (germination) and vegetative growth. However, other possible explanations exist, and further research is needed to better understand the differences between the different plant PAPS. Although much is now known about the cisacting RNA elements involved in mRNA 3 ’ end formation in plants [8], little progress has been made in understanding the biochemistry of this process. The present studies indicate that, like the polyadenylation signals themselves, the enzymes involved in mRNA polyadenylation in plants are probably quite different from their mammalian and yeast counterparts. Further study of the pea PAP should broaden our understanding of polyadenylation in plants, especially as more is learned about the modulation of its activity in vitro and as molecular clones encoding the components of this enzyme become available.
J. Das Guptu el al. /Plant Science 110 (1995) 215-226
226
Acknowledgments We thank Brian Rymond and Martha Peterson for helpful suggestions and Carol Von Lanken for excellent technical assistance. We are especially grateful to Marco Kessler and Claire Moore for the gift of monoclonal antibodies specific for the yeast PAP.
Tissue-specific and light-regulated expression of a pea nuclear gene encoding the small subunit of ribulose-l,5bisphosphate carboxylase. EMBO J., 3 (1984) 1671-1679.
[lOI B.D.
IIll
Mogen, M.H. MacDonald, Cl. Leggewie and A.G. Hunt, Several distinct types of sequence elements are required for efficient mRNA 3’ end formation in a pea rbcS gene. Mol. Cell. Biol., I2 (1992) 5406-5414. K.M. Rose and S.T. Jacob, Nuclear poly(A) polymerase
[I21
from rat liver and a hepatoma. Comparison of properties, molecular weights and amino acid compositions. Eur. J. Biochem., 67 (1976) 11-21. H. Towbin, T. Staehelin and J. Gordon, Electrophoretic
References III
E. Wahle and W. Keller, The biochemistry of 3’-end cleavage and polyadenylation of messenger RNA precursors. Annu. Rev. B&hem., 61 (1992) 419-440. I21 E. Wahle, A novel poly(A)-binding protein acts as a specificity factor in the second phase of messenger RNA polyadenylation. Cell, 66 (1991) 759-768. of a mamma131 E. Wahle, Purification and characterization lian polyadenylate polymerase involved in the 3’ end processing of messenger RNA precursors. J. Biol. Chem., 266 (1991) 3131-3139. 141 T. Raabe, F.J. Bollum and J.L. Manley, Primary structure and expression of bovine poly(A) polymerase. Nature, 353 (1991) 229-234. 151 E. Wahle, Cl. Martin, E. Schlitz, and W. Keller, Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase. EMBO J., IO (1991) 4251-4257. 161 J. Lingner, 1. Radtke, E. Wahle and W. Keller, Purification and characterization of poly(A) polymerase from Saccharomyces cerevisiae. J. Biol. Chem., 266, (I991 ) 8741-8746. 171 J. Chen and C.L. Moore, Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA. Mol. Cell. Biol., I2 (1992) 3470-3481. I81 A.G. Hunt, Messenger RNA 3’ end formation in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol., 45 (1994) 47-60. A
G. Coruzzi,
R. Broglie,
C. Edwards
and N.-H. Chua,
transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76 (1979) 4350-4354. E.F. Fritsch and T. Maniatis, Molecular (131 J. Sambrook, Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor, NY, 1989. 1141 M.M. Kessler, A.M. Zhelkovsky, A. Skvorak and C.L. Moore, Monoclonal antibodies to yeast poly(A) polymerase (PAP) provide evidence for association of PAP with cleavage factor I. Biochemistry, 34 (1995) l750- 1759. Purification and propcr1151 Y. Tarui and T. Minamikawa, ties of a poly(A) polymerase from Vigna unguiculata. Plant Cell Physiol., 29 (1988) 835-842. Poly(A) polymerase from II61 Y. Tarui and T. Minamikawa, Vigna unguiculata seedlings: a bifunctional enzyme responsible for both poly(A)-polymerizing and poly(A)hydrolyzing activities. Eur. J. Biochem., 186 (1989) 591-596. of I171 D. Saluja, M. Mathur and R.C. Sachar, Purification poly(A) polymerase from mung bean hypocotyls: subunit structure, molecular properties and characterization of the reaction product. Plant Sci., 60 (1989) 27-38. II81 R. Kapoor, N. Verma, D. Saluja, S. Lakhani and R.C. Sachar, Purification and characterization of poly(A) polymerase from germinated wheat embryos: enzyme glycosylation. Plant Sci., 89 (1993) 167-176.
ELSEVIER
Characterization
of a novel plant poly(A) polymerase
Jaydip Das Gupta, Qingshun Li, A. Brian Thomson, Arthur G. Hunt* Plant Physiology/Biochemistry/Molecular Biology Program, Department of Agronomy, Universiry of Kentucky. Lexington, KY 40564-0091. USA
Received 13 March 1995; revision received 8 June 1995; accepted 10 July 1995
We have purified and characterized poly(A) polymerases (PAPS) from Pisum sativum, Brassica juncea, and Zea mays. Through chromatography on DEAE-Sepharose and heparin-Sepharose, these PAPS copuritied as a single enzyme along with RNPs that could provide RNA substrates for the enzyme. More extensive purification by chromatography on MonoQ resulted in the resolution of the PAPS into as many as three fractions. One of these (PAP-l) contained a 43-kDa polypeptide immunologically related to the yeast PAP, and two others (PAP-II and PAP-III) contained RNAs that could serve as substrates for polyadenylation. These fractions by themselves possessed little PAP activity, but mix-
tures containing combinations of these displayed substantial activity. Similar PAP factors (PAP-l and PAP-III) were identified after fractionation of extracts prepared from Brassica juncea and Zea mays. The factors from one plant were completely interchangeable with those from different plants. We conclude that the poly(A) polymerases present in vegetative plant tissues consist of more than one component. In this respect, they are substantially different from other reported plant, mammalian, and yeast PAPS. Keywords: mRNA Polyadenylation;
RNA processing; Posttranscriptional
1. Introdnctioo
Messenger RNA 3 ’ end formation in eucaryotes is an RNA processing event involving an endonucleolytic cleavage at the so-called polyadenylation site, followed by the addition of a polyadenylate tract to the 3 ’ end of the processed RNA [ 11. In mammals, pre-mRNAs are processed by a set of factors that recognize a polyadenylation
* Corresponding author, Tel.: (+1-606) 257 3637; Fax: (+l606) 323 1952.
signal (AAUAAA) located upstream from the cleavage/polyadenylation site, and a UG-rich element located downstream from this site. These factors mediate the site-specific cleavage of the pre-mRNA and subsequent polyadenylation of the cleaved RNA. The polyadenylation reaction is catalyzed by a PAP and proceeds in two stages: the first entails the addition of about 10 nts, and requires the polyadenylation signal AAUAAA in the substrate RNA molecule; the second results in the addition of some 200 nts and involves, in addition to the PAP, a nuclear polyadenylate binding protein [2].
0168-9452/95/$0X50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(95)04205-Z
control
Characteristic PAPS are responsible for the addition of the polyadenylate tracts to the 3’ ends of mRNAs in mammalian and yeast cells The purified calf thymus enzyme consists of a mixture of polypeptides ranging between 57 and 60 kDa [3]; this protein is the product of a gene capable of encoding polypeptides of 77 [4] or 82 kDa [5], and the size of the protein purified from calf thymus has been attributed to proteolysis during purification [4]. This enzyme can polyadenylate RNAs non-specifically (e.g. independent of a polyadenylation signal) in the presence of Mn’+ [3] and acts in concert with other factors to process and polyadenylate precursor mRNAs in a poly(A) signal-dependent manner [l]. The purified yeast enzyme is a 63-kDa polypeptide that can also polyadenylate RNAs non-specifically [6]; with this enzyme, Mn*+ and Mg*+ can satisfy the divalent metal cation requirement. The yeast enzyme is one of several factors required for poly(A) signaldependent polyadenylation of precursor mRNAs
[71. Plant polyadenylation signals differ substantially from mammalian signals. In plant genes, a complex series of cis elements in a pre-mRNA is needed for efficient mRNA 3 ’ end formation [B]; these include elements that may be functionally analogous to the mammalian polyadenylation signal AAUAAA (termed near-upstream elements, or NUEs, in plant genes), the cleavage/polyadenylation site (CS) itself, and one or more novel elements (far-upstream elements, or FUEs) located upstream from the putative AAUAAA-like motifs. To better understand the process of mRNA 3 ’ end formation in plants, we have initiated biochemical studies focusing on possible polyadenylationrelated activities. Here, we describe the characterization of PAPS from three different plants. These enzymes can be chromatographically resolved into several fractions. One of these contains a polypeptide that is immunologically related to the yeast PAP; however, this fraction is not capable of polyadenylating exogenously added RNA. The other fractions contain populations of RNPs whose RNAs can serve as substrates for the PAP. Our studies indicate that the plant PAPS can be resolved into more than one component, and are thus different from PAPS isolated from other organisms.
2. Methods 2.1. Plant materials Garden pea (Pisum sativum cv. Laxton Progress) seed was obtained from Kentucky Garden Supply (Lexington KY). Seeds were sown in the greenhouse and harvested after 2-3 weeks of growth, when plants had two to three extended open trifoliates. Corn (Zea mays, Pioneer 524) was obtained from Dr Michael Barrett (Department of Agronomy, University of Kentucky); seeds were sown in the greenhouse and harvested after 8 days of growth. Indian mustard (Brassica juncea) was obtained from Dr R.K. Mandal (Department of Biochemistry, Bose Institute, Calcutta, India); seeds were sown in the greenhouse and harvested after 10 days. 2.2. RNA substrates Poly(A), average length - 400 nts, was obtained from ICN Biochemicals. To produce RNAs containing the wild-type rbcS-E9 polyadenylation signal, nts +703 through +1002 of the pea rbcS-E9 gene [9] were cloned as an NsiI-HaeIII fragment into PstI+EcoRV-digested Bluescript. Transcription using this plasmid (rbcS-E9-wt) as template yielded RNAs containing the complete rbcS-E9 polyadenylation signal [lo]; these RNAs are denoted as rbcS-EPwt RNAs in this paper. One additional RNA (termed ‘precleaved’, because it contains all of the sequences that should be present in a pre-mRNA that has been cleaved at a polyadenylation site but has not yet been polyadenylated) was prepared directly from a PCR-amplified template. Fifty nanograms each of two primers (5 ‘-TAATACGACTCACTATAGGGGTTTCGACAACGTTCGTCA-3’ and 5’TGAGAATGAACAAAAGGACC-3 ‘), approximately 50 ng of a plasmid containing the wildtype rbcS-E9 polyadenylation signal (pGB 1B) [B], 5 ~1 of 10 x Taq DNA polymerase buffer (1 x = 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgC12 and 0.01% gelatin) were brought to a volume of 50 ~1 with Hz0 and Taq DNA polymerase (0.2 units) was added. DNAs corresponding to nts -215 to +l of the rbcSE9 polyadenylation signal were amplified (35 cycles, each consisting of 1 min at 92°C 1 min at 45°C. and 2 min at 72”(Z), the PCR products were ex-
J. Das Gupta et al. /Plant Science II0 (199.0 215-226
tended at 72°C for 5 min and then purified by electrophoresis in agarose gels and elution from ion exchange paper. Approximately 1 fig of PCR product was used in each transcription reaction, as described above. Each RNA was used as described in the text for PAP reactions. Additionally, these were tested for ability to serve as substrates for the yeast PAP; in all cases examined, the in vitro transcripts proved to be suitable RNA primers for polyadenylation using the yeast enzyme (data not shown). 2.3. Poly(A) polymerase reactions The non-specific PAP assay was a modification of that described by Rose and Jacob [l I]. Unless otherwise indicated, the fraction of interest (5-20 ~1)was brought to a total volume of 50 ~1with buffer I (40 mM KCl, 25 mM Hepes-KOH pH 7.9, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10% glycerol and 5 pg/ml of each of leupeptin, chymostatin, and antipain). This was mixed with 9 ~1of PAP reaction mix (167 mM Tris-HCl pH 8.0, 267 mM KCl, 3.33 mM MgCI,, 0.33’mM EDTA, 3.33 mM DTT, 0.67% nonidet-P40, 1.28 mg/ml bovine serum albumin, 3.33 mM ATP, and l-2 rCi of [CY-3ZP]ATP). When indicated, poly(A) was added to a final concentration of 3.33 mg/ml and unlabeled &S-derived RNAs to a final concentration of 2.5 PM. Reactions were carried out at 30°C for 2 h and then stopped by extraction with phenol/chloroform/isoamyl alcohol (25:24: 1, v/v/v). Of the aqueous phase, 20 ~1 were spotted on a l-cm* piece of DE-81 filter paper (Whatman), the filters washed five times in 5% Na2HP04, and the incorporated radioactivity was measured by liquid scintillation spectrometry. In some instances, the products of polyadenylation reactions were analyzed on sequencing gels. The aqueous phases of the terminated reactions were precipitated with ethanol and the nucleic acids dried and analyzed on 6% sequencing gels. Gels were dried and visualized by autoradiography using Kodak XAR-5 film. 2.4. Analysis of endogenous RNAs Endogenous RNAs present in the various PAP preparations were isolated by extraction with phenol/chloroform/isoamyl alcohol and precipitation with ethanol. These RNAs were end-labeled
217
with purified yeast PAP (US Biochemicals) using the protocol suggested by the manufacturer. Briefly, 1 pg of RNA was incubated in a 30-~1 reaction containing 6 ~1 of the supplier’s 5 x reaction buffer (100 mM Tris-HCI pH 7.0, 250 mM KCI, 3.5 mM MnC12, 1 mM EDTA, 500 &ml acetylated bovine serum albumin, and 50% glycerol), l-2 &i of [ar-32P]ATP (1500-3000 Ci/mmol), and 100 units of yeast PAP. After 30 min at 30°C the reactions were terminated by extraction with phenol/chloroformIisoamyl alcohol, and nucleic acids recovered by alcohol precipitation. These were then separated on 6% sequencing gels. Gels were dried and visualized by autoradiography using Kodak XAR-5 film. Under these conditions, very short (20 nts or less) oligoadenylate tracts are typically added by the yeast PAP [6]. 2.5. RNAse A digestions and alkali treatment of nucleic acids To treat the products of polyadenylation by the pea and yeast enzymes with RNAse A, the RNAs recovered from a single reaction (typically, between lo4 and lo5 counts/min) were dissolved in 50 ~1 of 10 mM Tris-HCl pH 8.0 + 1 mM EDTA (TE), 5 ~1 of RNAse A (40 units/pi, from US Biochemicals) were added, and reactions carried out at 37°C for 30 min. Reactions were terminated by extraction with phenol/chloroform/isoamyl alcohol, tRNA was added to a concentration 100 &ml, and the nucleic acids recovered by alcohol precipitation and analyzed on sequencing gels as described above. To treat the products of the pea PAP reactions with alkali, the RNAs recovered from a single reaction were dissolved in 20 ~1 of TE as described in the preceding paragraph. Five microliters of this were mixed with 20 ~1 of TE and 25 ~1 of 2 N NaOH. After 30 min at room temperature, this solution was extracted with phenol/chloroform/ isoamyl alcohol (saturated with Tris-HCl pH 7.9), tRNA was added to a concentration of 100 pg/ml, and the nucleic acids recovered by alcohol precipitation and analyzed on sequencing gels as described above. 2.6. Purification of plant PAPS PAPS were isolated from green leaves of young plants. Unless otherwise indicated, all operations
218
J. Das Gupta CI (11./ Plunt Science
were done at 4°C. Leaves were harvested, chopped to tine pieces, and placed in grinding buffer (110 mM KCl, 15 mM Hepes-KOH pH 7.9, 5 mM MgCl*, 0.1 mM EDTA, I mM DTT, 1 mM PMSF, 9.4% w/v ammonium sulfate and 5 pglrnl of each of the protease inhibitors leupeptin, chymostatin, and antipain; 2 ml of buffer was used for each gram of leaf material). Leaves were then homogenized for 30 s using a Polytron tissue homogenizer (with 0.1% octanol as an antifoaming agent), the homogenate filtered through four layers of cheese-cloth, and the filtrate centrifuged at 7000 x g for 30 min. Solid ammonium sulfate was added to bring the supernatant to 35%. The solution was stirred for 30 min and centrifuged at 2000 x g for 10 min. Solid ammonium sulfate was then added to bring the supernatant to 70% saturation and the solution was stirred for 30 min and centrifuged as before. The resulting pellet was resuspended in buffer I. The extract was then dialyzed for 12- 16 h against a buffer (dialysis buffer) containing 40 mM KCl, 25 mM Hepes-KOH pH 7.9, 0.1 mM EDTA, 1 mM PMSF, 10% glycerol, and 12 mM fi-mercaptoethanol. After dialysis, the extract was stored at -80°C. A total of 500 mg of protein was loaded onto a 70-ml DEAE-Sepharose column (Sigma) that had been equilibrated with buffer I. The column was washed at 1 ml/min with 100 ml of buffer II (buffer I containing 200 mM instead of 40 mM KCI) and proteins that were retained on the matrix eluted with a IOO-ml gradient (200-500 mM) of KCI in buffer I. Fractions of 5 ml were collected and assayed for PAP activity without dialysis. Fractions containing active PAP were pooled and dialyzed against dialysis buffer. A total of 25 mg of protein was then loaded onto a 15-ml heparinSepharose (Pharmacia) column that had been equilibrated with buffer I. The column was washed with 35 ml of buffer I and 5-ml fractions collected. Proteins that were retained on the matrix were eluted with 50 ml of buffer III (buffer I containing 500 mM instead of 40 mM KC]). Fractions of 6 ml were collected and assayed for PAP activity without dialysis. PAP-containing fractions were stored at -80°C. The heparin-Sepharose flowthrough, which had a majority of the PAP activity, was loaded at 0.25
II0
( 1995) 215-226
ml/min onto a Mono-Q HR5/5 column (Pharmacia) that had been equilibrated with buffer I. This column was developed with a 20-ml linear gradient of KC1 (40-500 mM) in buffer I and l-ml fractions collected. Fractions were assayed for activity without dialyzing, and active fractions corresponding to PAP-I, -11, and -III (see Section 3, Results) were pooled and dialyzed against dialysis buffer. At this stage, the different PAP fractions had protein concentrations between 50 and 250 pg/ml. The PAP fractions were divided into 100~~1 aliquots and stored at -80°C. For further purification, 500 ~1 (approximately 10 pg) of Mono-Q purified PAP-I were separated, without prior dialysis, on Superose 6 columns. The sample was loaded, and the column developed, with buffer I at a flow rate of 0.25 ml/min. Onemilliliter fractions were collected and assayed for activity or analyzed by SDS-PAGE. 2.7. Micrococcal nuclease treatment of PAP-III A total of 100 ~1of PAP-III (after MonoQ chromatography) was brought to 5.6 mM in CaCIz by adding 11.2 ~1 of a 50 mM stock solution. Micrococcal nuclease (6 ~1 of a 15 I-J/~1stock solution) was added and the mixture incubated at 30°C for 30 min. A total of 14 ~1 of 50 mM EGTA (pH 7.0) was added to stop the micrococcal nuclease digestion and 20 ~1 of this mixture used directly in the PAP assay. For this treatment, controls in which the micrococcal nuclease was replaced with water were done as well. Treatments of labeled RNAs in the buffers used here indicated that micrococcal nuclease was active in these conditions and was capable of hydrolyzing >90% of input labeled RNAs. In some cases, micrococcal nuclease-treated PAP-III was extracted with phenol/chloroform/ isoamyl alcohol and the residual nucleic acids precipitated with ethanol and characterized as described above. 2.8. hmunoblot analysis For Western blots, proteins were separated by SDS-PAGE and the separated proteins transferred to a nitrocellulose membrane [ 121 using a TransBlot Cell (Bio-Rad Laboratories) following the manufacturer’s recommendations. Gels were
J. Das Gupta et al, /Plant
either stained with Coomassie Brilliant Blue [13] or probed with monoclonal antibodies that recognize an epitope in the C-terminal portion of the yeast PAP [14]. For the latter, filters containing transferred proteins were incubated (at room temperature) in TTBS for 5 min, TTBS containing 5% (w/v) nonfat dried milk for 1 h, and then with 5% milk-TTBS containing a 1:5000 dilution of monoclonal antibody for 12-16 h. The monoclonal antibody was removed with three washes (5 mm each) of TTBS, the filters incubated for 3 h with 5% milk-TTBS containing goat anti-mouse IgG alkaline phosphatase conjugate (Sigma), and this antibody conjugate removed with three washes of TTBS. Filters were equilibrated in alkaline phosphatase buffer (0.1 M Tris-HCl pH 9.5, 0.1 M NaCl, 0.05 M MgCl,) and developed in alkaline phosphatase buffer + 2 nitroblue mg/ml tetrazolium + 1.2 mg/ml 5-bromo4chloro-3indoyl phosphate; development was stopped by washing with deionized water.
Science I10 (1995) 215-226
219
crude extract 0.2 M
+-I
DEAE-Sepharose
ca. 0.35 M DEAE-PAP 0.5 M
heparin-Sepharose 0.04 M
I+ HS-PAP
MOIIOQ 0.25 M
0.36 M
0.43 M
4 PAP-I
4 PAP-II
4 PAP-III
Fig. 1. Illustration of the purification strategy for the pea PAP.
1.2 flfl
3. Results 1.0
3.1. Purification and characterization of a pea PAP It is difficult to measure PAP activity in crude pea extracts by following the modification of labeled input RNAs, presumably due to the presence of large quantities of non-specific nuclease activity in these extracts (B.D. Mogen and A.G. Hunt, unpublished observations). We therefore set out to characterize PAPS in these extracts using a nonspecific polymerization assay [l I]. This assay measures the incorporation of [or-32P]ATP into products that can be immobilized on ion exchange paper and is well suited measuring PAP activity in the presence of nucleases. Using this assay, potential PAP activity could be detected in crude extracts prepared from pea seedlings. Consequently, fractions containing possible PAP activity were obtained by chromatography on DEAESepharose and heparin8epharose (summarized in Fig. 1). The active fractions after heparin8epharose chromatography were by all accounts a poly(A) polymerase. In particular, this activity was inhibited by 3’-dATP (Fig. 2), presumably as a result of chain termination. Moreover, polymeriz-
0.2 0.0 pdy(A)
+
Mg2+ Mn2+ 3’-dATP
+
+
-
-
-
+
-
+
I
+
-
+
-
+
-
+
I
-
-
+
-
-
+
Fig. 2. Properties of the HS-PAP. Reactions were performed as described in Section 2, Methods, with the exceptions noted beneath the graph: poly(A) was added to some reactions to a final concentration of 3.33 mg/ml, MgCI, was replaced with MnCl* (0.5 mM), or ATP was replaced with a mixture of unlabeled 3’-dATP (0.5 mM) + 1-2 &i of [~Y-~~P]ATPper reaction. Reactions contained IO ~1 (5.4 fig) of HS-PAP and were incubated at 30°C for 2 h before extraction and quantitation.
J. Das Cupta et al. /Plant Science I10 (1995) 215-226
220
ing activity was not apparent when [CX-“PI3‘dATP, [w~~P]CTP, [a- 32P]GTP, or [w~~P]UTP were used as nucleotide substrates (not shown). This activity, termed the HS-PAP, displayed a marked preference for Mg2+ over Mn2+ (Fig. 2) and had an apparent KM for ATP of 39 f 1.5 PM in the presence of 0.5 mM Mg2+. However, in these studies, addition of poly(A) (Fig. 2) or rbcSEPderived RNAs (data not shown) had little effect on PAP activity; indeed, the HS-PAP was remarkably active in the absence of added RNA (Fig. 2). The activity in the absence of exogenously added RNA suggests the presence of endogenous RNA in the HS-PAP. However, other possible reactions could account for the production of labeled products able to bind to ion exchange paper. Thus, to assure that the labeled products we measured were the products of a polyadenylation reaction, the products obtained from reactions
lacking exogenous added RNA were characterized. After extraction with phenol and chloroform, the labeled products were analyzed on sequencing gels (Fig. 3). A population of large nucleic acids was evident after autoradiography; these nucleic acids were susceptible to RNAse A (Fig. 3A) and alkali (Fig. 3B), indicating that they were RNA. Moreover, after RNAse A digestion, these products had a broad size distribution (Fig. 3A). Since RNAse A does not digest polyadenylate tracts, this result indicates the presence of extended poly(A) tails in the molecules labeled by the HSPAP. These data rule out the presence of any activities that might initiate poly(A) formation de novo (as these would not be affected by treatment with RNAse A) and suggest that the HS-PAP contains an endogenous population of RNAs that can serve as substrates for this enzyme. Further attempts to purify the pea PAP consistently resulted in a dramatic loss of activity.
B.
A. RN Ase
Ar:
-
+
alkali:
-
+
364
242
Fig. 3. Sequencing gel analysis of RNAs produced by HS-PAP. Polyadenylation reactions were performed with 10 ~1 (5.4 pg) of HSPAP, [w~*P]ATP and no added RNA and were incubated at 30°C for 2 h. The labeled products isolated by phenol/chloroform extraction and ethanol precipitation. (A) One sample was analyzed on a sequencing gel before and one after RNAse A digestion. Positionsof DNA size standards are shown on the left. (B) One sample was analyzed on a sequencing gel before and one after treatment with alkali. Positions of DNA size standards are shown on the left.
J. Das Gupta et al. /Plant Science I10 (199.5)215-226
After MonoQ chromatography, less than 5% of the activity loaded onto the column was recovered. One possible explanation for this loss of activity was that the HS-PAP was actually a multicomponent enzyme, the individual components of which were resolved by chromatography on MonoQ. To test this, we systematically assayed each MonoQ fraction for activities that stimulated the PAP recovered from this column. In this manner, three components, designated as PAP-I, PAP-II, and PAP-III in respective order of their elution from the MonoQ column, could be identified (Fig. 1). Interestingly, the activity of the reconstituted enzyme at this stage of purification was still largely independent of exogenously-added RNA, indicating the presence of endogenous RNAs in one or more components. In some preparations, all three components proved to be required for optimal activity (Table 1, Experiment 1). In others, however, maximal activity could be observed with just the combination of PAP-I + PAP-III (Table 1, Experiment 2). For this reason, further experiments focused on PAP-I and PAP-III. 3.2. PAP-I contains a polypeptide that is immunologically related to the yeast PAP
The above studies indicate that the PAP activity in pea extracts is capable of utilizing RNAs present in endogenous RNPs, but they do not identify which of the three PAP components includes the
221
catalytic moiety responsible for actual polyadenylation. In an attempt to determine this, each component was analyzed by immunoblotting using a monoclonal antibody raised against the yeast PAP. This experiment revealed the presence, in PAP-I, of a 43-kDa polypeptide that was recognized by the monoclonal antibody (Fig. 4B). Equally abundant polypeptides in PAP-II and -111 (Fig. 4A) were not recognized (Fig. 4B), and omission of the monoclonal antibody from the procedure eliminated the appearance of the band seen in Fig. 4B (data not shown). These results indicate a specific interaction between the 43-kDa polypeptide and the antibody raised against the yeast PAP. This 43-kDa polypeptide was consistently seen in several (> 10) different Mono-Q PAP-I preparations, and was one of two polypeptides that coactivity on chromatographed with PAP-I Superose-6 columns (Fig. 5). Taken together, the results presented in Figs. 4 and 5 suggest that the 43-kDa polypeptide seen in PAP-I is analogous to the yeast PAP. However, PAP-I was unable to polyadenylate exogenously added RNAs (Table 2), including RNAs that contain an intact polyadenylation signal (rbcS-E9-wt) and RNAs that resemble the putative products of pre-mRNA cleavage by the polyadenylation ap-
Table I Reconstitution using PAP-I, II and III Enzyme
Experiment I
Experiment 2
HS PAP PAP-I PAP-II PAP-III PAP-1 + II PAP-I + III PAP-II + III PAP-I + II + III
35 894 176 1077 1224 2529 20 378 19094 63411
ND 0 121 504 429 12 137 4428 9580
Enzyme reactions were performed as described in Section 2, Methods, with IO ~1 (5.4 rg) of HS-PAP or with 5 ~1 of PAP-I, PAP-II and PAP-III, as indicated. The results of two experiments, done with different preparations of PAP, are shown as counts/mm retained on DE-81 paper, corrected for background. ND, not determined.
Fig. 4. SDS-PAGE and immunoblot analysis of PAP-I, -II and -III. Aliquots of each PAP component were separated on duplicate 12% acrylamide gels containing SDS. One gel (A) was stained with Coomassie Brilliant Blue [I31 and the other (B) probed with a monoclonal antibody specific for the yeast PAP [l4]. The immunoreactive band in part B corresponds in size to the principal stainable band in PAP-1 in part A.
222
J. Das Gupta et al. /Plant
PAP activity fraction
5.
21
20
19
18
Science 110 (19%)
17
16
.?l.f-226
15
g
M 218
102 72 43 29 18
Fig. 5. Superose 6 purification of PAP-I. PAP-I obtained after Mono-Q chromatography was further purified by chromatography on Superose 6. Aliquots (40 ~1) of each fraction were assayed for PAP-I activity in the presence of PAP-III (10 al). Alternatively, aliquots (100 cl) were separated on polyacrylamide gels containing SDS. which were subsequently stained with Coomassie Brilliant Blue 1131.The polypeptide that is recognized by monoclonal antibodies raised against the yeast
Table 2 PAP-I cannot polyadenylate exogenously added RNAs RNA
Counts/min
None Poly(A) rbcS-E9-wt rbcS-E9-PC PAP-III
0 7 0 0 13 28l=
Reactions were performed as described in Section 2. Methods, with IO ~1 of PAP-I. PAP-III (IO ~1) or the indicated RNAs were added to the respective reactions and PAP activity determined as described in Section 2, Materials and methods. Results are given as counts/mm retained on DE-81 paper, corrected for background. ‘In the absence of PAP-I, PAP-III incorporated a net of 186 counts/mm into poly(A).
PAP (Fig. 48) is indicated by the arrow on the left and the sizes of molecular weight standards on the right. The column fractions are denoted above their corresponding lanes, as are lanes containing Mono Q-purified PAP-I (onto) and molecular weight size standards (M). The PAP activity measured after mixing each fraction with PAP-III is depicted above the lane designations; for reference, the activities in fractions 17, 18, and 19 were 33 165, 46 477 and 7660 counts/min, respectively.
paratus (rbcS-E9-PC). Thus, although PAP-I contains a polypeptide that is immunologically related to the yeast PAP, it is not itself the sole component of the corresponding plant enzyme. Instead, additional factors present in PAP-III are required for activity.
3.3. PAP-III contains endogenous RNAs As was the case with the HS-PAP, the reconsitution of PAP activity with MonoQ-purified components did not require added RNA (Table 1). This observation suggested the presence of RNA in one or more of these components. Accordingly, aliquots of PAP-I, -11, and -111were extracted with phenol/chloroform extraction and possible nucleic acids recovered by ethanol precipitation. About 50 ng of nucleic acid per c(g of protein could be
J. Das Gupta et al. /Plant
recovered from MonoQ-purified PAP-II, and 250 ng of nucleic acid per pg of protein from PAP-III. In contrast, no nucleic acid (< 10 rig/g of protein) could be recovered from PAP-I. The nucleic acids recovered from PAP-III had a broad size distribution (Fig. 6, lane 1). In some preparations, as shown in Fig. 6, these nucleic acids were relatively small, ranging between 20 and 150 nts. In other preparations, nucleic acids as large as 400 nts could be observed (not shown). These observations indicate that the nucleic acids present in PAP-III are heterogeneous in terms of size. These nucleic acids were susceptible to digestion with RNAse A (Fig. 6, lanes 2 and 4). Therefore, PAPIII contains a population of RNAs with a broad range of sizes. This result confirms the suggestion regarding endogenous RNAs that was inferred from the experiment shown in Fig. 3. Similar results were obtained with preparations of PAP-II (data not shown). The RNAs present in PAP-II and PAP-III survive an extraction and purification process that exposes them to considerable endogenous nuclease activity. This suggests an association with one or more proteins in a ribonucleoprotein (RNP) complex. To examine this possibility, PAP-III was treated with micrococcal nuclease, and the treated samples examined for nucleic acid content and for PAP activity in the presence of PAP-I and absence of added RNA. Treatment of PAP-III with micrococcal nuclease had no discernible effect on the size distribution of RNAs present in PAP-III (Fig. 6, compare lanes 1 and 3). Moreover, the combination of PAP-I and micrococcal nucleasetreated PAP-III retained 77% of the activity of the combination of PAP-I and untreated PAP-III (not shown). Therefore, the endogenous RNAs in PAPIII were largely resistant to micrococcal nuclease. This result indicates the presence of a population of RNPs in PAP-III, the RNAs of which can serve as substrates for the reconstituted PAP. 3.4. Other plants have PAP-I and PAP-III that can substitute far the pea PAP-I and PAP-III me fractionation of the pea PAP into at least two components is an unusual characteristic not seen with PAPS from other organisms. Moreover, pips from other plants have been described, en-
Science 110 (1995)
215-226
223
1234 147 118
67
27
Fig. 6. Characterization of nucleic acids present in PAP-III. RNA was isolated from PAP-III before (lanes I and 2) or after (lanes 3 and 4) treatment with micrococcal nuclease and endlabeled with purified yeast PAP. Shown are the products of labeling using the yeast PAP and [3ZP]ATP, before (lanes I and 3) and after (lanes 2 and 4) RNAse A digestion. Positions of DNA size standards are shown on the left. The labeled molecules remaining after RNAse A digestion are less than IO nts in size, and their low abundance indicates that the majority of label seen in the lane without RNAse A digestion was incorporated as very short oligoadenylate tracts, tracts that were not recovered by ethanol precipitation following RNAse A treatment.
zymes that can be purified as single polypeptides [15-181. It was thus possible that the PAP we purified was one unique to peas, for any of a number of reasons. To examine this possibility, we fractionated extracts prepared from Brassica juncea and Zea mays. Chromatographic profiles of PAP activity in these extracts mirrored those seen with pea extracts: PAP activity bound to DEAE columns and was present in the flow-through of
J. Das Gupta el al. /Plan! Science 110 (1995) 215-226
224
125
El corn PAP-III El mustard PAP-III pea PAP-III 25
none
corn
mustard
pea
PAP-I Fig. 7. Interchangeability of PAP-I and PAP-III isolated from different plants. PAP-I and PAP-III was isolated from maize, mustard and pea as described in the text. Aliquots of IO pl were tested for activity in the combinations shown. The activity of each PAP-III preparation in the absence of PAP-I (none) was compared with the activities measured after combining with the maize, mustard and pea PAP-I. Reactions were performed as described in Section 2, Methods (note that no exogenous RNA was added in these reactions). The activity of each PAP-I preparation in the absence of PAP-III was indistinguishable from background. The data shown were corrected for background (- 350 counttimin)
columns (data not shown). Moreover, these preparations utilized endogenous RNAs as substrates, much as did the pea enzyme. Further fractionation of the PAPS from these other plants by chromatography on MonoQ also yielded results similar to those seen with the pea enzyme. For each enzyme preparation, components with chromatographic properties similar to PAP-I and PAP-III could be seen. These separated components by themselves had little PAP activity (with or without exogenously added RNA), but when combined yielded substantial activity (Fig. 7). Moreover, the respective components were completely interchangeable: PAP-I from all plants was able to reconsitute PAP activity with the PAP-III from peas, etc. (Fig. 7). Therefore, the three plants examined all possess a functionally equivalent PAP, one that can be chromatographically resolved into more than one component.
heparin-Sepharose
4. Discussion We have characterized a PAP from several plants that is distinctive in many respects. It can be resolved after chromatography into at least two components. One of these (PAP-I) has no inherent PAP activity but consists of a polypeptide that is immunolgically related to the yeast PAP (Fig. 4) and copurifies with PAP-I activity (Fig. 5). Another component (PAP-III) has a low inherent activity that is markedly stimulated by PAP-I (Table 1). This component also includes a population of RNPs (Fig. 6), the RNAs of which can serve as substrates for the reconstituted PAP (Figs. 2, 3). The fact that different plant PAPS can be resolved into more than one component, each of which is required for activity, distinguishes these enzymes from other reported PAPS [3,6,15- 181. It is possible that this result is a consequence of protease action during the purification process, action that
J. Das Gupta et al. /Plant Science I IO (1995) 215-226
cleaves a larger PAP into two smaller polypeptides. The observation that PAP-I and PAP-III from different plants are functionally interchangeable (Fig. 7) argues against this possibility, as this would require the presence of a relatively specific, conserved proteinase in the three plant species studied here. However, we cannot completely rule out the action of a proteinase as an explanation of the multi-component nature of the PAPS we have studied. PAP-III contains a significant quantity of RNA (Fig. 6) that can serve as substrates for polyadenylation (Figs. 2, 3; Tables I, 2). These RNAs survive exposure to endogenous nucleases and treatment of PAP-III with micrococcal nuclease (Fig. 6, lane 3), indicative of their residing in an RNP. This feature of PAP-III raises two interesting possibilities regarding the nature of plant PAPS. It is possible that PAP-III includes, along with these RNPs, an additional polyadenylation factor that copurities with the RNPs (perhaps one that is derived from the action of proteases, as suggested above). Thus, the combination of this factor and PAP-I would be required for PAP activity, and the RNAs present in PAP-III would serve as convenient, available substrates for the reconstituted enzyme. Alternatively, it is possible these plant PAPS consist of an enzyme (PAP-I) and an associated group of RNPs (PAP-III) that serve as the preferred or sole RNA substrates for PAP-I. Further purification and the cloning of genes encoding different polypeptide components of PAP-I and PAP-III will be needed to completely distinguish between these possibilities. In most purifications, an additional component (PAP-II) can be identified (Table 1). This component has readily detectable inherent activity and contains substantial RNA. We presume that PAPII is a combination of PAP-I and PAP-III, and its chromatographic behavior on MonoQ is consistent with this presumption. However, we cannot rule out other explanations for the properties of this component, and further study will be needed to better understand this component. PAPS from a number of different plants have been described by others. Tarui and Minamikawa purified a PAP from germinated cowpea seeds [15,16]. This enzyme consisted of a 63-kDa poly-
225
peptide [IS] that had both PAP and poly(A)hydrolyzing activity [16]. PAPS from mung bean [ 171and wheat [ 181have been identified and found to consist of single polypeptides with molecular weights ranging of 30 kDa (for the mung bean enzyme) or 70 kDa (for the wheat enzyme). All the plant enzymes identified to date displayed a complete dependence upon exogenously added RNA and a marked preference for Mn2+ over Mg2+. The enzymes we describe here have no detectable poly(A) hydrolyzing activity (unpublished results), prefer Mg2+ over Mn2+, and copurify with a population of endogenous RNPs that can serve as the RNA substrate for PAP activity. It is thus likely that the enzymes we have characterized are different from the PAPS from cowpea, mung bean, and wheat germ. In the latter two respects, the enzymes we have characterized also differ from the purified mammalian and yeast enzymes [3,6]. The reasons for the differences between the PAPS we describe here and plant PAPS described by others are not known. Different PAPS may be present at different developmental stages in the growth of plants. The PAPS described here were isolated from leaves of plants that had been grown for l-3 weeks, whereas the PAPS identified in other plants were purified from embryonic tissues. The differences between the PAPS we describe and other plant PAPS may thus be due to the involvement of different PAPS in early development (germination) and vegetative growth. However, other possible explanations exist, and further research is needed to better understand the differences between the different plant PAPS. Although much is now known about the cisacting RNA elements involved in mRNA 3 ’ end formation in plants [8], little progress has been made in understanding the biochemistry of this process. The present studies indicate that, like the polyadenylation signals themselves, the enzymes involved in mRNA polyadenylation in plants are probably quite different from their mammalian and yeast counterparts. Further study of the pea PAP should broaden our understanding of polyadenylation in plants, especially as more is learned about the modulation of its activity in vitro and as molecular clones encoding the components of this enzyme become available.
J. Das Guptu el al. /Plant Science 110 (1995) 215-226
226
Acknowledgments We thank Brian Rymond and Martha Peterson for helpful suggestions and Carol Von Lanken for excellent technical assistance. We are especially grateful to Marco Kessler and Claire Moore for the gift of monoclonal antibodies specific for the yeast PAP.
Tissue-specific and light-regulated expression of a pea nuclear gene encoding the small subunit of ribulose-l,5bisphosphate carboxylase. EMBO J., 3 (1984) 1671-1679.
[lOI B.D.
IIll
Mogen, M.H. MacDonald, Cl. Leggewie and A.G. Hunt, Several distinct types of sequence elements are required for efficient mRNA 3’ end formation in a pea rbcS gene. Mol. Cell. Biol., I2 (1992) 5406-5414. K.M. Rose and S.T. Jacob, Nuclear poly(A) polymerase
[I21
from rat liver and a hepatoma. Comparison of properties, molecular weights and amino acid compositions. Eur. J. Biochem., 67 (1976) 11-21. H. Towbin, T. Staehelin and J. Gordon, Electrophoretic
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