Limited Tryptic Digestion of Messenger RNA Capping Enzyme from Artemia salina ISOLATION OF DOMAINS FOR GUANYLYLTRANSFERASE AND RNA

The partially purified preparation of messenger RNA guanylyltransferase from Artemia salina contains, as in the case of the rat liver enzyme (Yagi, y., Mizumoto, K., and Kaziro, Y. (1983) EMBO J. 2,611-615), the RNA 5‘-triphosphatase activity which spe- cifically removes the y-phosphoryl group from the 5’-triphosphoryl end of the newly synthesized mRNA molecule. The enzyme consists of a single polypeptide chain of M, = 73,000 and forms a covalent enzyme-GMP complex as an intermediate for the guanylyl- transferase reaction. Upon limited hydrolysis with trypsin, the en~yrne-[~’P]GMP complex is converted to a smaller 32P-containing fragment of M, = 44,000. When the free enzyme, not complexed with GMP, is digested with trypsin under the same condition as above, the digests retain almost full activities of both guanylyltransferase and RNA 5‘-triphosphatase and can form an en~yme-[~’P]GMP complex of the size of M, = 44,000 on incubation with Functional domains harboring the activities of guanylyl- transferase and RNA 5”triphosphatase are separated by gel filtration on a Sephacryl

The capping enzyme (mRNA guanylyltransferase) has been purified from vaccinia virus (1) and various cellular sources, including rat liver ( 2 ) , HeLa cells (3-5), wheat germ (6), calf thymus (7), and mouse myeloma cells (5). Previous studies have revealed that the capping reaction occurs on the 5'diphosphoryl end of the nascent mRNA as shown in the following equation (2). rPLV P,(uJ guanylyltransferase PPPG " PPN-aptoll ' GpppN-+ !6i * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Therefore, y-phosphoryl group of the primary transcript has to be removed before transguanylylation takes place. The presence of an RNA 5"triphosphatase activity has been reported in purified vaccinia virus capping enzyme complex (Mr = 127,000) (8,9). More recently, we have shown that the guanylyltransferase purified from rat liver, consisting of a single polypeptide chain of M , = 69,000, contains an RNA 5 'triphosphatase activity which specifically removes the yphosphoryl group from the triphosphate-terminated polyribonucleotide (10).
In this paper, we report that guanylyltransferase from Artemia salina is also a multifunctional enzyme possessing both guanylyltransferase and RNA 5"triphosphatase activities in a single polypeptide chain. Furthermore, the two domains retaining the catalytic activities are separated by limited proteolysis with trypsin and purified by ion exchange column chromatographies.

EXPERIMENTAL PROCEDURES
Materiak-[u-32P]GTP and sodium [3ZP]pyrophosphate were purchased from Amersham Japan and New England Nuclear, respectively. Bacterial alkaline phosphatase and nuclease P1 were obtained from Worthington Biochemical Co. and Yamasa Shoyu, Choshi, respectively. Bovine pancreas trypsin and soybean trypsin inhibitor were the products of Sigma. A. salina eggs (encysted embryos) were obtained from Aquarium Stock Co., New York.
RNA Substrate~-5'-[r-~~P]ATP-terminated poly(A) was prepared as previously described (10) with a slight modification as follows. Cordicepin (3'-deoxyadenosine)triphosphate was added to the reaction mixture at a concentration of 10 /IM (ATP/cordicepin triphosphate, 601) to decrease the chain length of poly (A) and to increase the incorporation of [ T -~~P I A T P .
Purification of Guanylyltransferase-Guanylyltransferase from A. salina was partially purified from encysted embryos. Detailed purification procedure will be described elsewhere. Crude extracts (100,000 X g supernatant fraction) were prepared as described (11) and fractionated with ammonium sulfate between 40 and 80% saturation. After dialysis against 20 mM Tris-HC1 (pH 7.9), 0.1 mM EDTA, 5 mM 2-mercaptoethanol, 0.5 mM Mg(OAc)Z and 20% glycerol (Buffer A) containing 50 mM KC1, the proteins were chromatographed on a CM-Sephadex column with a linear gradient of KC1 (60-300 mM) in Buffer A. The guanylyltransferase activity was eluted at about 0.22 M KC1 as a single peak. The active fraction was concentrated with ammonium sulfate and chromatographed on a DEAE-Sephadex column with a linear gradient of KC1 (50-300 mM) in Buffer A. Approximately 170-fold purification was obtained with a recovery of 12% starting from the crude extracts. The preparation did not contain any GppppG synthetase activity (12).

RESULTS
Association of RNA 5'-Triphosphatase with Guanylyltrans-/erase-When the ammonium sulfate fraction (40-80% saturation) of the A. salina crude extract was chromatographed on a CM-Sephadex column with a linear gradient of KCl, the activities of guanylyltransferase and RNA 5"triphosphatase were co-eluted at 0.22 M KC1 and well separated from the activity of RNA (guanine-7-)methyltransferase which was eluted a t 0.18 M KC1 (data not shown). The active fractions were combined and further purified on a DEAE-Sephadex J" ?- Guanylyltransferase fractions (22.8 mg of protein) from the CM-Sephadex column were concentrated by ammonium sulfate precipitation and applied to a column (0.8 X 30 cm) of DEAE-Sephadex. The proteins were eluted with a 200-ml linear gradient from 50 to 300 mM KC1 in Buffer A. Fractions of 1.5 ml were collected and assayed for activities of the en~yme-[~'P]GMP complex formation (O), GTP-PPi exchange (0). and RNA 5"triphosphatase ( X ) using 3-, 3-, and 0.2-p1 aliquots, respectively, of each fraction.

B.
C. GTP and treated for 30 min at 0 "C without protease ( l a n e I ) , with trypsin (protein/trypsin, 45:l (w/w)) ( l a n e 2), or with chymotrypsin (p!otein/chymotrypsin, 45:l (w/w)) ( l a n e 3). After incubation, protelns were precipitated with cold trichloroacetic acid, washed with ether, and subjected to electrophoresis in a 10% polyacrylamide gel in the presence of sodium dodecyl sulfate. B, the enzyme-["PIGMP complex was incubated with trypsin (protein/trypsin, 60:l (w/w)) at 0°C for 0 ( l a n e I ) , 5 ( l a n e 2). and 10 min ( l a n e 3). Proteins were precipitated and electrophoresed as in A. C, the native enzyme not reacted with [n-"PIGTP was treated with trypsin (protein/trypsin, 701 (w/w)) at 0 "C for 0 ( l a n e I ) , 5 ( l a n e 2 ) , and 10 min ( l a n e 3), after which time trypsin inhibitor (inhibitor/trypsin, 3:l (w/w)) was added to terminate the reaction, and then each sample was incubated with [n-"PIGTP and electrophoresed as in A. Ori, origin.  (1.7 mg of protein) was applied to a column of Sephacryl S-200 (0.7 X 50 cm) equilibrated with Buffer A containing 100 mM KC1 and was eluted with the same buffer. Fractions of 0.2 ml were collected and assayed for enzyme. GMP formation (0) and RNA 5"triphosphatase ( X ) using 20-and 5-p1 aliquots, respectively. B, elution profile of the trypsinized guanylyltransferase. The enzyme preparation (2.1 mg of protein) was incubated with trypsin (protein/trypsin, 701 (w/w)) for 10 min at 0°C. After the addition of trypsin inhibitor (inhibitor/trypsin, 3:l (w/w)). the digest was loaded on the same column of Sephacryl S-200 and eluted as above. Fractions (0.2 ml) were collected and assayed for the capping activity (O), enzyme-GMP complex formation (O), and RNA 5'-triphosphatase (X), using 5-, 8-, and 1-pl aliquots, respectively. BSA, bovine serum albumin; Tu, Escherchia coli elongation factor Tu; Cyt c, cytochrome c. column. As shown in Fig. 1, RNA 5'-triphosphatase activity was eluted again in parallel to the activities of guanylyltransferase assayed by formation of the enzyme. GMP complex and GTP-PPi exchange.
The molecular weight of the A. salina guanylyltransferase-["PIGMP complex was estimated to be 73,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (see Fig.   2A, lane I). Since the molecular weight of the native enzyme was 74,000 as determined by gel filtration (see Fig. 3A), we concluded that A. salina guanylyltransferase is also a multifunctional enzyme consisting of a single polypeptide chain and catalyzing two different reactions as in the case of rat liver guanylyltransferase (10).
Limited Proteolysis of Guunylyltransferase-The guanylylt r a n s f e r a~e -[~~P ] G M P complex was incubated with trypsin or chymotrypsin (protein/protease, 45:l (w/w)) for 30 min at 0 "C and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As shown in Fig. 2A, the 73-kDa enzyme-["PIGMP complex was quantitatively converted to a new single band of 44 kDa by digestion with trypsin ( l a n e 2). On the other hand, chymotrypsin was less active than trypsin and the enzyme was only partially converted to a fragment of 45 kDa (lane 3 ) . Fig. 2B shows the time course of trypsin digestion of the enzyme-[:"P]GMP complex. Almost all the complex was converted to a 44-kDa fragment after incubation for 10 min a t a protein/trypsin ratio of 60. To examine whether or not the 44-kDa fragment generated by limited proteolysis is catalytically active, we first treated guanylyltransferase with trypsin for various times a t a protein/trypsin ratio of 70 and then analyzed for the activity of the digested enzyme to form a pr~tein-[''~P]GMP complex. As shown in Fig. 2C, a 44-kDa fragment-[:'2P]GMP complex was formed when the material which had been treated with trypsin for 5 min was incubated with [n-:"P]GTP (lune 2). It was also found that the RNA 5"triphosphatase activity was not impaired, but rather stimulated to about 1.7-fold (data not shown).
Isolation of Domains for Guanylyltransferase and RNA 5'-Triphosphatase-When the enzyme was digested with trypsin and chromatographed on a Sephacryl S-200 column, the activities of guanylyltransferase and RNA 5"triphosphatase were eluted as two separate peaks a t positions corresponding to 44 and 20 kDa, respectively (Fig. 3 B ) . On the other hand, in the case of the undigested enzyme, both activities were eluted at the position of 74 kDa (Fig. 3A).
Complete separation of two domains was achieved by ion exchange chromatography as shown in Fig. 4. When trypsinized guanylyltransferase was chromatographed on a CM-Sephadex column with a linear gradient of KCl, RNA 5'triphosphatase and guanylyltransferase activities were separated completely and eluted a t 0.05 and 0.18 M KCl, respectively. The fraction of RNA 5"triphosphatase released %'P as inorganic phosphate from [y-""P]pppA(pA), but not from [y-:'2P]ATP, indicating that the RNA 5"triphosphatase domain still retains the same substrate specificity as the native enzyme. The purified guanylyltransferase domain was active only when the ppG-terminated RNA, but not the pppGterminated RNA, was used as "cap" acceptor molecules. This is in contrast to the native enzyme which can transfer GMP to either pppG-or ppG-terminated RNA to form "capped" GpppG-RNA. Only after incubation with RNA 5"triphosphatase domain, triphosphate-terminated RNA (pppA-RNA) could serve as a substrate for the purified 44-kDa guanylyltransferase domain (Fig. 5). After incubation, RNA was treated with nuclease P1 and alkaline phosphatase and electrophoresed on Whatman DE81 paper at pH 3.4 as described (2).
In a previous paper (IO), we have reported that RNA guanylyltransferase purified from rat liver nuclei contains the activity of RNA 5"triphosphatase. In this paper, we presented evidence that RNA guanylyltransferase purified from A. salina is also a multifunctional enzyme possessing both RNA guanylyltransferase and RNA 5"triphosphatase activities in a single polypeptide chain. After tryptic digestion, both activities were fully retained but the enzyme was cleaved into two fragments which were separated on a Sephacryl S-200 column to yield a 44-kDA guanylyltransferase domain and 20-kDa RNA 5"triphosphatase domain. Both domains were completely separated by CM-Sephadex column chromatography (Fig. 4).
Similar domain structures also seem to be present in rat liver capping enzyme, since the 69-kDa enzyme-["PIGMP complex was converted to a 40-kDa complex by the partial digestion with trypsin. Itoh et al. (13) reported that the size of the yeast guanylyltransferase-['"PIGMP complex in sodium dodecyl sulfate-gel electrophoresis is 45 kDa, the value which is similar to that of the guanylyltransferase domain of A. salina and rat liver. The yeast enzyme-GMP complex was refractory to trypsin digestion. Since the yeast capping enzyme consists of two small (39-kDa) and two large (45-kDa) Separation of Two Catalytic Domains of mRNA Capping Enzyme subunits,' we assume that, in yeast, guanylyltransferase and RNA 5'-triphosphatase may exist in two different subunits.
From these observations it seems that the cellular guanylyltransferase activity resides in a 40-to 45-kDa polypeptide.
On the other hand, vaccinia virus guanylyltransferase-[32P] GMP (95 kDa) was converted to a 60-kDa fragment by treatment with trypsin under similar conditions. More recently, we succeeded in purifying both domains to almost homogeneous states. Studies to further characterize the two domain structures are now in progress.