Eur. J. Biochem. 176,431 -439 (1988) 0 FEBS 1988

Purification of RNA 3’-terminal phosphate cyclase from HeLa cells Covalent modification of the enzyme with different nucleotides

Oscar VICENTE and Witold FILIPOWICZ Friedrich Miescher-Institut, Basel

(Received March 10/May 27, 1988) - EJB 88 0290

RNA 3’-terminal phosphate cyclase has been purified about 6000-fold to near homogeneity from HeLa cells. The purified protein is a single polypeptide with an M , of 38000-40000 and a Stokes radius of 2.66 nm. The cyclase shows a pH optimum of 8.0-9.0. In the presence of Mg2+ and ATP this enzyme catalyzes the conversion of a 3’-phosphate group into the cyclic 2’,3’-phosphodiester at the 3‘ end of RNA, through formation of a covalent cyclase-AMP intermediate. GTP, CTP and UTP (but not dATP or ADP) can also function as cofactors in the cyclization reaction, although less efficiently (apparent K,,, values for ATP and GTP are 6 pM and 200 pM, respectively). Consistent with this, the enzyme can be covalently labelled with the four [u-~~PINTPs.

Nucleoside cyclic 2‘,3’-phosphates and cyclic-phosphate- terminated oligonucleotides are generated as the intermedi- ates of RNA cleavage, catalyzed by cyclizing ribonucleases (reviewed in [l , 21). A possible anabolic function of the cyclic 2‘,3‘-phosphates in RNA emerged when it was found that eukaryotic RNA ligases require 2’,3’-cyclic ends for RNA ligation [3 - 51. This requirement applies to each of the two distinct eukaryotic RNA ligases described so far: (I) the RNA ligase active in plants and lower eukaryotes, catalyzing the formation of a 2’-phosphomonoester, 3’,5’-phosphodiester linkage by ligation of 2’,3‘-cyclophosphate and 5‘-phosphate (or 5’-hydroxyl) ends [3,4, 6- 121; (11) the RNA ligase active in animal cells which joins 2’,3’-cyclophosphate and 5’-hy- droxyl ends via the normal 3’,5’-phosphodiester bond [5, 131. Both RNA ligases seem to be involved in the tRNA splicing process [9- 11, 14- 161; the plant ligase is also the most likely candidate for the enzyme responsible for the generation of circular viroid and virusoid RNAs from their linear precursors

Another finding related to the function of 2‘,3‘-cyclic phos- phate in RNA metabolism in eukaryotes was the discovery of RNA 3’-terminal phosphate cyclase. This enzyme, catalyzing the ATP-dependent conversion of the 3’-phosphate group to the cyclic-2‘,3’-phosphodiester at the end of RNA, was first identified in HeLa cells and Xenopus oocytes [5]. The cyclase was subsequently partially purified from HeLa cell extracts and a mechanism of cyclization of the 3‘-phosphate has been proposed [20, 211. The physiological function of the RNA 3’- phosphate cyclase is not known. Initially it was suggested [5, 20,211 that the cyclase could be responsible for the generation of the 2’,3’-cyclic termini required for ligation of RNA by

[17- 191.

Correspondence to W. Filipowicz, Friedrich Miescher-Institut, P. 0. Box 2543, CH-4002 Basel, Switzerland

Abbreviations. PMSF, phenylmethylsulphonyl fluoride; pCp, cytidine 3’,5’-bisphosphate; 8, 32P-labelled phosphate group; > p, 2’,3’-monophosphate.

Enzymes. RNase T1 (EC 3.1.27.3); RNase T2 (EC 3.1.27.1); nuclease PI (EC 3.1.30.1); calf intestine alkaline phosphatase (EC 3.1.3.1); T4 RNA ligase(EC 6.5.1.3); trypsin (EC 3.4.21.4); pro- teinase K (EC 3.4.21.14).

eukaryotic ligases and, consequently, be involved in tRNA splicing. It was found, however, that splicing endonucleases responsible for the excision of introns from tRNA precursors in yeast [22] and Xenopus [23], directly produce RNA mol- ecules with cyclic phosphate ends. Although these findings do not necessarily rule out the involvement of the RNA cyclase in tRNA processing (e.g. the enzyme could be responsible for the regeneration of cyclic ends in the 3’-phosphate-terminated tRNA halves, formed by the action of decyclizing phos- phodiesterases), they make this possibility rather unlikely.

As a step in our investigation of the structure and function of the RNA 3’-terminal phosphate cyclase, we describe below the purification of the enzyme from HeLa cells. Partial characterization of the protein and its activation by different ribonucleoside triphosphates are also reported.

MATERIALS AND METHODS

Materials and reagents

Calf intestine phosphatase and nuclease P1 were obtained from Boehringer, RNases TI and T2 from Calbiochem and T4 RNA ligase from Pharmacia. All nucleoside mono- and diphosphates were obtained from Sigma, ATP and GTP from Boehringer, and CTP and UTP from Pharmacia. Protease inhibitors (benzamidine, aprotinin, bacitracin, leupeptin, PMSF) were purchased from Sigma. Poly(ethyleneglyco1) 20000 and Bio-Gel concentrator resin were Fluka and Bio- Rad products, respectively. Radioisotopes were from New England Nuclear. Heparin-Sepharose, poly(A)-Sepharose, blue Sepharose and prepacked FPLC Mono-S columns were provided by Pharmacia. DEAE-cellulose (DE-52) was from Whatman and TLC cellulose plates from Merck.

HeLa cell cultures

HeLa cells were grown in suspension cultures, in Joklik- modified Eagle’s medium (Gibco) containing 5% new born calf serum (Flow), to a density of 5 x lo5 cells/ml. The cells were washed twice with phosphate-buffered saline, frozen in solid COz and stored at - 70°C.

432

Purification of R N A 3’-terminal phosphate cyclase

All operations, except loading and running of the Mono-S column (step 5 , performed at room temperature), were carried out at 0-4°C. Unless indicated otherwise, the samples were frozen in solid COz after each step and stored at - 70 “C. The following buffers were used: buffer A, containing 20 mM Hepes/KOH, pH 7.6, 0.1 mM EDTA, 0.5 mM dithiothreitol, 10% (by vol.) glycerol; buffer B, same as buffer A but contain- ing also the protease inhibitors (0.1 mM benzamidine, 0.2 pg/ ml aprotinin, 10 pg/ml bacitracin, 0.2 pg/ml leupeptin, 0.01 mM PMSF) and 0.01% (by vol.) Triton X-100; buffer C, same as buffer A but containing also 0.01 mM PMSF and 0.01% Triton X-100. The three initial steps of purification were similar to those described previously [20].

Step I . 100000 x g supernatant. 100 ml of packed HeLa cells (3.3 x lo1’ cells, from 65 1 of suspension cultures) were thawed and a lOOOOOxg supernatant was prepared as de- scribed by Weil et al. [24]. The extract (270 ml, 497 mg protein) was extensively dialyzed against buffer A containing 75 mM NaCl.

Step 2. DEAE-cellulose. 125 ml of packed wet DEAE- cellulose (DE-52), equilibrated in the above buffer, was added to the 100000 x g supernatant and the suspension was gently stirred for 3 h. Protein not retained was collected by filtration, the cellulose cake was washed with an additional 100 ml buffer and both filtrates were combined (344 ml, 241 mg protein).

Step 3. Heparin-Sepharose. The material was applied to a heparin-Sepharose CL-6B column (2.5 x 16 cm), equilibrated in buffer A containing 75 mM NaC1. The column was washed with 350 ml of the same buffer and retained protein eluted in two steps with buffer A containing 200mM and 450mM NaC1. No cyclase activity was detected in the flow-through and 200 mM NaCl fractions. The fraction eluted with buffer A containing 450 mM NaCl was extensively dialyzed against buffer A containing 75 mM NaCl(27 ml, 38 mg protein).

Step 4 . Poly(A)-Sephnrose. A column of poly(A)- Sepharose 4B (1.6 x 10 cm) was packed and equilibrated in buffer B containing 75 mM NaCl; 60 ml bovine serum albu- min solution (0.2 mg/ml) in the same buffer, was then applied to the column, in order to saturate unspecific binding sites on the poly(A)-Sepharose. The albumin was eluted with buffer B containing 600 mM NaCl and the column reequilibrated in buffer B containing 75 mM NaC1. The heparin-Sepharose fraction was then applied and the column washed with buffer B containing 150 mM NaCl. Retained protein was eluted with a linear gradient of 150 - 600 mM NaCl in buffer B, at 18 ml/ h, collecting 2-ml fractions. The fractions with highest cyclase activity, eluting at about 0.5 M NaCl (see also [21]), were pooled (42 ml, 0.45 mg protein), concentrated fivefold in a dialysis bag by dehydration against dry poly(ethyleneglyco1) 20000, and extensively dialyzed against buffer B containing 75 mM NaCl.

Step 5. Mono-S. Dialyzed protein from the previous step was directly applied to a Mono-S HR 5 /5 column, equilibrated in buffer B containing 75 mM NaC1. Th: column was washed with 15 ml of the same buffer and retained protein was eluted with 17 ml of a linear gradient of 75-350 mM NaCl in buffer B at a flow rate of 1 ml/min. Fractions with cyclase activity, eluting at 250 mM NaCl, were pooled (3.3 ml, 50 pg protein).

Step 6. Blue-Sepharose. A blue-Sepharose CL-6B column (0.9 x 0.6 cm) was equilibrated in buffer C containing 75 mM NaC1. 2 ml of the albumin solution (0.2 mg/ml) in the same buffer was first applied and the column washed with buffer C containing 2 M NaCl. The column was re-equilibrated with

buffer C containing 75 mM NaCl, and the Mono-S fraction, diluted to 8 ml with buffer C (without NaCl), was then ap- plied. The column was washed with 10 ml of the buffer and retained protein was eluted stepwise with buffer C containing 0.2 M, 0.6 M, 1 M and 2 M NaCl. Most of the recovered cyclase activity (z 70%) was eluted at 0.6 M NaCl (fraction BS-600); these fractions were pooled (3.5 ml, 3.5 pg protein) and stored frozen at - 70 “C in small aliquots. Protein eluting at 1 M NaCl (fraction BS-1000) contained about 30% of the recovered cyclase activity and was pooled separately.

Glycerol gradient centrifugation

0.5 ml of the blue-Sepharose (BS-600) fraction was con- centrated about tenfold in a dialysis bag, by dehydration against Bio-Gel concentrator resin, dialyzed against buffer C containing 75 mM NaCl and loaded onto a linear gradint of 15-35% glycerol (3.9 ml) in the same buffer. After centrifugation for 36 h at 56000 rpm in a Beckman SW-60 rotor, fractions of 100 pl were collected, assayed for cyclase activity (see Fig. 2) and the material in fractions 16-21 was pooled.

Preparation of radioactive substrate

A mixture of (Np),,G3’$ oligoribonucleotides (n = 3 to 73) was prepared as follows. Unlabelled tobacco mosaic virus RNA was extensively digested with RNase T1 and the digest treated with phosphatase to remove the 3’-terminal phos- phates [25]. The oligonucleotide mixture was incubated with [5’-32P]pCp (6Cp) and T4 RNA ligase, to produce (Np).Gfl Cp [25]. The reaction mixture (1 5 pl) contained 70 mM Hepes/ KOH, pH 8.3, 10 mM MgClZ, 3 mM dithiothreitol, 10% glycerol, 10% dimethylsulfoxide, 40 pM ATP, 12 pM flCp (spec. act. 1200 Ci/mmol), 4.6 pg oligonucleotide mixture and 7 units T4 RNA ligase. After 14 h at 4”C, the sample was diluted with 4 vol. 50 mM Hepes/KOH, pH 7.6, containing 3 mM EDTA, and incubated at 37°C for 30 min with 5 units RNase T1; SDS was then added to a final concentration of 0.1% and the sample extracted with phenol/chloroform/ isoamyl alcohol (25: 24: 1, by vol.) mixture. The aqueous phase was applied to a 10-ml Sephadex G-25 (fine) column, equilibrated and eluted with 20 mM ammonium aceate, pH 5.5. The peak of radioactivity corresponding to 32P- labelled oligonucleotides was collected, lyophilized and dis- solved in HzO.

The radioactive substrates were analyzed by digestion with RNase T2, nuclease P1 or phosphatase, followed by TLC on cellulose plates in solvents A and/or B [3]. Between 90 - 97% of the label, depending on the preparation, was present in the 3’-terminal G3’p.

R N A 3’-terminal phosphate cyclase assay

Cyclase activity was assayed by measuring the amount of charcoal (Norit)-adsorbable, phosphatase-resistant 32P radio- activity generated after incubation of cyclase fractions with the (Np),,Gfi substrate. Unless indicated otherwise, reaction mixtures (10 pl) contained, including contribution of the cyclase fraction, 30 mM Hepes/KOH, pH 7.6, 187.5 mM NaCl, 4 mM MgClZ, 0.15 mM EDTA, 0.1 mM spermidine, 1.25 mM dithiothreitol, 5% glycerol, 0.3 mM ATP, 0.005% Triton X-100, 0.005 mM PMSF, 150-250 fmol radioactive substrate and the amounts of cyclase as indicated. After 20 min at 25”C, 1 p1 10% SDS and 1 unit phosphatase were

433

added and incubation was continued for 30min at 37°C. Reactions were stopped by dilution with 0.5 ml 20 mM HCl containing 1 % sodium phosphate and addition of 0.2 ml Norit suspension (25% Norit in 80 mM HCl). After 10 min on ice, the charcoal was collected by filtration on glass- fiber GFjC filters and radioactivity determined by Cerenkov counting with 32% efficiency. Blank values (assays without RNA cyclase added) were substracted. When protease sensi- tivity of cyclase was measured, the enzyme preparation was extensively dialyzed against buffer A containing 75 mM NaCl and 0.01% Triton X-100 in order to eliminate PMSF. One unit of cyclase activity is defined as the amount of protein that renders 1 pmol of 32P-labelled termini of (Np).G$ resistant to phosphatase under the standard assay conditions.

Apparent K , values for ATP and GTP were calculated from Lineweaver-Burk plots. Assays were performed under the standard conditions, in the presence of five different con- centrations of ATP (1-9 pM) or GTP (12.5-200 pM). All velocities were calculated from the initial linear rates.

SDSjpolyacrylamide gel electrophoresis

The 10% or 15% polyacrylamide gels were run as de- scribed by Laemmli [26]. To the protein samples, after boiling in the presence of 2% SDS and 10mM dithiothreitol, iodoacetamide was added to 40 mM concentration and incu- bation continued for 30min at 22°C in the dark [27]. The silver-staining procedure was according to Biirk et al. [28].

Labelling of R N A cyclase with [ E - ~ ~ P I N T P s and analysis of the labelled nucleotides complexed with cyclase

Cyclase fractions were incubated with [E-~~PINTP in a final volume of 45 - 90 pl under standard cyclase assay con- ditions, except that (Np).GI*) substrate was omitted. The concentration and specific activity of ATP or the other nucleoside triphosphates are indicated in the legends. After incubation at 25°C for the indicated times, the products of the reaction were analyzed by SDS/polyacrylamide gel electrophoresis and autoradiography. In the experiments shown in Fig. 6, samples were supplemented with the corre- sponding unlabelled NTP (final concentration 10 mM) im- mediately before being applied to the gel. To identify the nucleotides complexed with cyclase, silver-stained gels were extensively washed with water after autoradiography and the radioactive bands corresponding to cyclase were cut out. Polyacrylamide pieces were fragmented and boiled for 5 min

in 0.2 ml 0.2 M HC1. Samples were filtered through glass- wool in an Eppendorf tip, washed with water, concentrated 100-fold by evaporation and analyzed by cellulose TLC in solvent A or B. Autoradiography of gels and TLC plates was performed at - 70 "C with intensifying screens.

Protein determination

In all fractions, with the exception of blue-Sepharose (step 6), protein concentration was measured by the method of Bradford [29], using the Bio-Rad reagent and bovine serum albumin as standard. Protein concentration of the BS-600 fraction was determined by fluorimetry after reaction with o-phthalaldehyde [30] using the Mono-S cyclase fraction as standard.

Thin-layer chromatography

Nucleotides were separated on TLC cellulose plates in solvent A [saturated (NH4)2S04/1 M sodium acetate/isopro- panol(80: 18: 2, by vol.)] or solvent B [t-butanol/concentrated HCljH20 (14: 3: 3, by VO~.)].

RESULTS

Purification of cyclase

The RNA 3'-terminal phosphate cyclase has been purified from HeLa cells more than 6000-fold, to near homogeneity. A summary of the purification procedure, described in detail in Materials and Methods, is presented in Table 1 and Fig. 1. The previous finding that the enzyme can be covalently labelled with [E~~P]ATP [20, 211 (and data presented below) greatly facilitated the identification of the cyclase polypeptide during the final steps of purification. The 40-kDa protein band comigrating in SDS/polyacrylamide gels with the [E-~~PIATP- labelled cyclase appeared to be already considerably enriched after poly(A)-Sepharose and Mono-S chromatography (Fig. IA, lanes 4 and 5) . Further purification revealed how- ever that only a fraction of the 40-kDa material corresponded to the RNA 3'-phosphate cyclase. Chromatography on the blue-Sepharose column permitted the separation of the cyclase from the accompanying protein of identical molecular mass. Cyclase activity eluted from this column at 0.6 M and 1 M NaCl concentration (fractions BS-600 and BS-1000, re- spectively), while the contaminating 40-kDa polypeptide (and another major contaminating protein of 36 kDa) could only

Table 1. Purification of RNA 3'-terrninal phosphate cyclase One unit o f cyclase activity corresponds to the amount of protein that renders 1 pmol labelled termini of (Np),Gfi resistant to phosphatase under standard assay conditions

Purification step Total protein Specific activity Total activity Purification Yield

1. I00000 x g supernatant 2. DEAE-cellulose 3. Heparin-Sepharose 4. Poly(A)-Sepharose 5. Mono-S 6. Blue-Sepharose

BS-600 BS-1000

mg 497 24 1

38 0.45 0.05

0.0035" -

uni ts/mg 170 265

1120 60833

205 882

1025000 -

units -fold %

84490 1 100 63865 1.6 76 42 560 6.6 50 27375 358 32 10294 1213 12

3 588 1600

a Protein concentration determined by the phthalaldehyde method (see Materials and Methods).

434

Fig. 1 . SDS 10% 1~0/~,[/[,/.~/(//?7idc, gtJ/ e/r.c~trophoresis 0 / cyc’1u.w ut d@wnt pur~ficmtion stugc~s. (A) The following fractions were analyred : lane 1. 0.5 pg 100000 x g supcrnatant; lane 2, 0.5 pg DEAE-cellulose fraction; lane 3, 0.5 pg heparin-Sepharose fraction; lane 4, 0.22 pg poly(A)- Scpharosc fraction; lane 5.0.22 pg Mono-S fraction; lane 6 , 0.06 pg blue-Sepharose (BS-600) fraction; lane 7, 15 pI of the blue-Sepharosc gcl taken from the column alter i t had been washed with buffer C containing 2 M NaCl (see Materials and Methods and Results) and subjected to the siinie treatment BS the other protein samples. Positions of marker proteins [phosphorylase h (92.5 kDa), bovine scruin albumin (66.2 kl>a). ovalbumin (45 k l h ) (Bio-Rad)] are indicated. (B, C) Polyacrylamide gel analysis of: lane 1 , Mono-S fraction (270 ng. 55 units cyclasc activity); lane 2. blue-Sepharose BS-600 fraction (43 ng, 44 units cyclasc). Prior to electrophoresis cyclase fractions wcrc incubated w i t h [r-3LI’]ATI’ (40 pM, spec. act. 4 Ci/mmol) for 2 h under standard conditions. A picture of the silver-stained gel is shown in B. and its autoradiogram ( 1 -day exposure) in C. The position of the cyclase is indicated by an arrow; asterisks mark contaminating polypcptidcs i n fraction 13s-600

10 20 30 40

Fraction number

Fig. 2. G / ~ w r o / grtrt/icv?/ c~c~ritrifugatio~7 of’ q d u s e BS-600 fruction. (A) Profile of cyclase activity. 0.5 pg BS-600 fraction was subjected to gradient sedimentation as described under Materials and Methods. 0.4 pI of the indicated fractions were assayed for cyclasc activity v. i t h 240 l‘inol (Np),,Gfi substrate. Positions of marker protcins [bovine serum albumin (BSA, 67 kDa), ovalbumin (43 kI>a). chymotrypsinogcci A (25 kDa) and ribonuclease A (13.7 kDa) (Pharmacia)], sedimented in identical parallel gradients, are indicated by arrows. (13. C ) SIIS 10‘%, polyacrylamide gel analysis of gradient fractions after labelling with [ z - ~ ~ P I A T P . 60 pl of glycerol gradient fractions 15 (lane 1 ) and 17 - 22 (lanes 2-7) were incubated with [z-~’P]ATP (40 pM, spec. act. 2 Ci/mmol) for 3 h under standard conditions; lane HS contained 20 111 (20 11g protein) o f RS-600 fraction that was not incubated with [X-~’P]ATP. Marker proteins are the same as in Fig. 1 A cxccpt lor tho addition of the soybean trypsin inhibitor (21.5 kDa). The band corresponding to cyclase is indicated by an arrow, and the contaminating prolcins in fr , ‘iction . ’ BS-600 by asterisks. (€3) Silver-stained gel; (C) autoradiogram (1 8-h exposure)

43 5

F: E X

2-

Phosphorylase b A

Cyclase Ovalbumin *,$

LO -

Trypsin inh 20 -

1 3 5 Mobil ity (cm)

Fraction number

B

Cyclase 4 0 Ovalbumin */

Cyclase 4 0 rA Ovalbumin

i Chymotrypsinoger

/ RNase

I I I

1 .o 1.5 2.0 Volume (ml)

D

I I L 4

0.2 0.3 0.4 0.5 Kav

80

T 40 2

X s 20

Fig. 3. Molecular mass determination of the purified cyclase. (A) Estimation of cyclase molecular mass by SDS/polyacrylamide gel electrophoresis. M , of marker proteins are plotted on a logarithmic scale against protein mobility; values are taken from a 15% polyacrylamide gel. The mobility of RNA cyclase is indicated by an arrow. Marker proteins are the same as in Fig. 2B. (B) Molecular mass estimation by glycerol gradient sedimentation. M , of marker proteins are plotted on a logarithmic scale against the sedimentation position of each protein. Marker proteins are as shown in Fig. 2A. (C, D) Molecular mass determination by gel filtration. 0.22 pg Mono-S fraction was subjected to gel filtration on a Sephacryl S-200 HR column (0.9 x 58 cm) equilibrated and eluted in buffer A containing 0.5 M NaCl and 0.01 YO Triton X- 100, at 12 ml/h. 0.5-ml fractions were collected and 2 gl of the indicated fractions were assayed for cyclase activity. (C) Profile of cyclase activity. Elution positions of marker proteins are indicated. Void volume (V,) was determined with blue dextran. (D) M , of marker proteins plotted on a logarithmic scale against K,, values of each protein. K,, of RNA cyclase is indicated by an arrow

be recovered and visualized when the blue-Sepharose gel, after being washed with 2 M NaCl, was boiled with SDS and the sample analyzed by electrophoresis (Fig. lA, lane 7). Thus, the possibility that the BS-600 fraction still contains the con- taminating 40-kDa protein seems unlikely. Moreover, when the BS-600 fraction was subjected to rechromatography on blue-Sepharose, no appreciable amounts of the 40-kDa poly- peptide were retained on the column (results not shown). The possibility that the 40-kDa contaminating protein represents a fraction of cyclase which binds strongly to the resin was eliminated by the results shown in Fig. 1 B and C. SDS/ polyacrylamide analysis of Mono-S and BS-600 protein samples containing similar amounts of cyclase units, the ac- tivity being measured either by cyclization of (NP)~G$ or by protein adenylation in the presence of [u-~~PJATP, revealed a substantial increase in specific activity of the 40-kDa band after the blue-Sepharose step.

Electrophoretic analysis of the BS-600 fraction, followed by silver staining and densitometric scanning, indicated that the 40-kDa cyclase band represents 76% of the protein applied (Fig. 1 A, lane 6); fraction BS-1000 showed a similar protein profile. An aliquots of the BS-600 fraction was further purified

by glycerol gradient centrifugation. A single symmetrical peak of activity was observed (Fig. 2A). Cyclase activity cosedi- mented with the 40-kDa band of adenylated protein as deter- mined by SDS/polyacrylamide gel analysis, followed by silver staining (Fig. 2B) and autoradiography (Fig. 2C). Gel electrophoresis of the gradient fractions (Fig. 2B) also indicat- ed that after glycerol gradient sedimentation the enzyme does not contain detectable amounts of the contaminating proteins (lanes 1-7) which were still present in the BS-600 fraction (lane BS). The artifactual bands visible in all gel lanes are caused by overstaining with silver.

Properties of the purified enzyme

Purified cyclase was stable for at least 6 months when stored at -7O"C, and activity was not affected by several cycles of freezing and thawing. The enzyme was highly sensi- tive to heating; preincubation of the glycerol gradient fraction for 5 min at 42°C or 50°C decreased cyclase activity by 55% and more than 95%, respectively. The cyclase was relatively resistant to protease digestion, a property observed with crude enzyme preparations [5,20]. Incubation of the BS-600 fraction

436

40 80 120 Protein (pg)

6 7 8 9 1 0 PH

20 LO 60 80 Time (minl

I I

2 L 6 Concentration (mM )

Fig. 4. Requirements of cyclase reaction. (A) Dependence on enzyme concentration. (B) Kinetics of the reaction at 0°C (0), 12°C (A) , 25°C ( O ) , 30°C (0) and 37°C (*). (C) pH optimum; the following buffers were used, at 40 mM concentration: Mes/NaOH (0), Mops/ NaOH (A) , Tris/HCl (O) , ethanolamine/HCl ( O ) , Hepes/KOH, pH 7.6 (*). (D) Divalent cation requirement; chloride salts were used, at the indicated concentrations. The BS-600 fraction was used in all experiments; samples in B, C and D contained 80 pg protein. Assays were performed as indicated in Materials and Methods, with 150 fmol (Np)"Gfl substrate. In experiment C, 100 mM Hepes/KOH, pH 7.9, was additionally included during incubations with phosphatase

with a 100-fold excess of proteinase K or trypsin for 20 min at 37°C inhibited attivity by only 34% and 48%, respectively (data not shown).

The cyclase (BS-600 fraction) did not contain detectable DNase activity (assayed with double- or single-stranded sub- strates) or RNase activity (assayed with a uniformly 32P- labelled SP6 polymerase transcript of the human /I-globin gene). The glycerol gradient fraction was free of detectable ATPase, GTPase and unspecific phosphatase activities (as- sayed with [Y-~~PIATP, [E-~~PIATP and [ U - ~ ~ P ] G T P as sub- strates), and of nucleoside diphosphokinase activity (assayed with [Y-~~PIATP and GDP).

Analysis of the purified cyclase on 10% (Fig. 2) or 15% (Fig. 3A) polyacrylamide gels in the presence of SDS indicat- ed an apparent M , = 40000. A sirnilair value (38000) has been obtained from glycerol gradient sedimentation (Fig. 3 B). These values are in agreement with previous M , determi- nations by the same methods, performed with partially purified enzyme [20,21]. However, gel filtration of the cyclase (Mono-S fraction) on a Sephacryl S-200 HR column, equili- brated in buffer A containing 75 mM NaCI, indicated an M , of 26000 (data not shown), in line with our previous observations [20]. The anomalous behaviour of the enzyme in

c ATP

Time ( h )

Fig. 5. Activity of differeni nucleotides in the cyclization reaction. Samples containing 0.25 p1 cyclase (fraction 18 from glycerol gradi- ent; see Fig. 2) and 250 fmol (Np).Gfi substrate were incubated under standard conditions in the presence of 0.25 mM ATP ( O ) , GTP (U), CTP (A) , UTP (V), ADP (*) or dATP (0), or in the absence of nucleotide (0)

the Sephacryl column could be due to ionic interactions with the gel matrix, resulting from the strongly basic character of the protein. To check this possibility, the cyclase was subjected to gel filtration on the same column, but in the presence of 0.5 M NaC1. The elution position of the enzyme in high salt indicated an M , of 38 000 (Fig. 3 C and D), in agreement with the values obtained by other methods. The Stokes radius of the cyclase, as estimated by the method of Ackers [31], was found to be 2.66 nm.

Reaction requirements

The amount of cyclized substrate was linearly dependent on enzyme concentration up to about 80 pg protein/lO p1 (Fig. 4A). The reaction proceeded linearly for 20 min in the experiment shown in Fig. 4B; when more substrate was avail- able the cyclization reaction remained linear for more than 1 h (not shown, see also Fig. 5). Under standard assay con- ditions the reaction proceeded until the (Np).Gfi substrate was almost quantitatively converted to the 2',3'-cyclic product (Fig. 5 and data not shown). The rates of reaction were similar at 25", 30" and 37°C; at 0" and 12°C the rates were 25% and 60% of the value found at 30°C, respectively (Fig. 4B). The reaction showed a broad pH optimum with a maximum be- tween pH 8 and 9 (Fig. 4C). Inclusion of 0.005% Triton X-100 in the reaction mixtures greatly increased the reproducibility of activity measurements, particularly when low amounts of purified cyclase were used.

The reaction required the presence of Mg2+ ions; optimal concentration of MgC12 was 4-5 mM. Only 5% of the ac- tivity was found when Mg2+ was replaced with Mn2+ or Ca2+ (Fig. 4D). Optimal concentration of monovalent ions (Na' or K') was 150-200 mM, with 50% activity observed at 50 mM and 400 mM NaCl or KC1. Preincubation with -SH group reagents like N-ethylmaleimide (1 mM, 10 min at 25 "C) or iodoacetamide (3 mM, 15 min at 25°C) completely inacti- vated the RNA 3'-phosphate cyclase (data not shown, see also [211).

437

Fig. 6. Analysis o / c:vc/use-NMP cornpkexes. (A) Labelling of RNA cyclase with different [x-~’P]NTPs. Samples containing 15 ng BS-600 fraction were incubated with 100 pM [u-~’P]ATP (lane l ) , [s(-”PIGTP (lane 2), [ X - ~ ~ P I C T P (lane 3) or [s(-”PIUTP (lane 4). Each samplc contained 100 pCi NTP (spec. act. 6.8 Ci/mmol). After incubation for 24 h under standard conditions, samples were analyzed on an SDS/ 10% polyacrylamide gel. (B) Phosphatase resistance of cyclase-NMP complexes. Samples of BS-600 fraction containing 25 ng protein wcrc incubated for 20 h under standard conditions with 100 pM [x-~’P]ATP (lane l ) , [ X - ~ ~ P ] G T P (lane 2), [x-~’P]CTP (lane 3) or [r-”P]UTP (lane 4). Specific activity of each NTP was 14 Ci/mmol. 0.2 units phosphatase was then added to each sample. After incubation for 90 min at 37 C. samples were analyzed by SDS/polyacrylamide gel electrophoresis. Autoradiograms of the wet silver-stained gels are shown (2-days exposure). CIP, calf intestinal phosphatase. (C) TLC analysis of labelled nucleotides complexed with cyclase. Radioactive bands corresponding to the cyclase were excised from the gel shown in B. The amount of radioactivity in lanes 1 - 4 was 436 cpm, 110 cpm, 162 cpm and 60 cpm (Cerenkov). respectively. Samples were processed as described in Materials and Methods and analyzed by TLC in solvent A. The lanes are marked as in B. Positions of marker nucleotides are indicated. The autoradiogram of the TLC plate is shown. Lane 1 was exposed for 3 days and lanes 2 - 4 for 10 days

effect o f ATP und other nucleotides

It was found previously that cyclization of the 3’-phos- phate in RNA. catalyzed by the cyclase, requires ATP as cofactor and that the cyclase can be covalently labelled with [Y-~’P]ATP. It appears that the adenylated enzyme is an inter- mediate in the cyclization process and that in the subsequent step of the reaction AMP is transferred from the protein to the 3’-phosphate of RNA, resulting in the formation of the activated -N3’pp5’A end [20, 211.

In the experiment shown in Fig. 5, the activity of cyclase (glycerol gradient fraction) was measured in the presence of either ATP or other nucleotides. The results indicate that UTP, CTP and GTP can act as cofactors, although in their presence the reaction proceeds 15 -20 times more slowly than in the presence of ATP. No activity was detected when dATP or ADP were used in place of ATP.

The apparent K , values for ATP and GTP (as an example of the other three nucleoside triphosphates) were calculated to correspond to 6 pM and 200 pM, respectively. The K , of 6 pM for ATP was also found by Reinberg et al. 1211. To rule out the possibility that the activity observed in the presence of GTP, CTP or UTP was due to low amounts of ATP con- taminating these nucleotides, or due to traces of ADP and nucleoside diphosphokinase present in the cyclase preparation (although nucleoside diphosphokinase activity was not detect- ed by direct measurements, see above), labelling of cyclase with different [cY-~’P]NTPs was studied. RNA cyclase was incubated in the presence of each of the four [a-32P]-labelled ribonucleoside triphosphates and the samples, after boiling in 2% SDS and the addition of an excess of the corresponding non-radioactive NTP, were analyzed by SDS/polyacrylamide gel electrophoresis. Radioactive bands with the mobility of cyclase were observed in all four cases (Fig. 6A). The labelling

with [ x - ~ ~ P ] U T P was considerably lower than with the other triphosphates although UTP showed similar activity as GTP or CTP when phosphate cyclization was measured (Fig. 5). The strong background observed in the lane containing labelled GTP and, to a lesser extent, in other lanes, is most likely due to cross-linking of low amounts of the nucleotides to the acrylamide matrix, in the presence of formaldehyde used for gel fixation and silver staining [28].

In the following experiment (Fig. 6 B), the complexes formed during the reaction of the cyclase with different [ U - ~ ~ P I N T P S were treated with phosphatase prior to gel electrophoresis. In all cases the radioactivity remained associ- ated with the 40-kDa cyclase band, supporting the conclusion that the nucleotides are covalently associated with the protein through the phosphate group. Treatment with phosphatase also eliminated the background in the gel lines containing [x-~’P]GTP, [LX-~’P]CTP and [ x - ~ ~ P I U T P (Fig. 6A). The labelled band of 70 kDa present in the four lanes corresponds to the phosphatase subunit which becomes phosphorylated during the reaction [32].

The measurement of the [E-~’P]ATP radioactivity associat- ed with the cyclase band indicated that the stoichiometry of nucleotide/RNA-cyclase varied in different experiments over 0.15-0.35 (data not shown). Assuming that one molecule of AMP is bound per molecule of cyclase these values indicate that not all purified protein is enzymatically active. The nucleotide/cyclase stoichiometry for GTP, CTP and UTP was lower than that for ATP (see legend to Fig. 6C). In order to determine directly the nature of the nucleotides complexed with the cyclase, the labelled 40-kDa bands were excised from the gel shown in Fig. 6B and the gel fragments were treated with 0.2 M HCI at IOO‘C for 5 min. This treatment should lead to the hydrolysis of the phosphoamide bond via which AMP 1211 (and presumably the other nucleotides) seems to be

438

covalently linked to the cyclase. The products of hydrolysis were analyzed by TLC on cellulose plates (Fig. 6C). In each case radioactive spots corresponding to the expected nucleoside 5’-phosphate could be visualized in the autoradiogram of the TLC plate. The major spot with high mobility seen in the analysis of [a-32P]GTP-labelled material (Fig. 6C, lane 2) is likely to correspond to the product of hydrolysis of the N-glycosidic bond in GMP; GMP is known to be more susceptible to acid hydrolysis than other ribonucleotides [33]. It should be stressed that no traces of radioactive AMP were detected in samples labelled with [E-~~PIGTP, [u-~’P]CTP or [E-~~PIUTP, which excludes the possibility that labelling of the cyclase with these nucleotides could be due to the formation of [U-~~PIATP during the initial reaction. Similar results were obtained when the nucleotides, released by acid treatment of the labelled cyclase bands seen in Fig. 6A, were analyzed by TLC (data not shown).

Altogether, these results demonstrate that the cyclase can form covalent complexes with all four ribonucleoside 5‘-phos- phates.

DISCUSSION

In this work we describe a procedure for the preparation of nearly homogeneous RNA 3’-terminal phosphate cyclase from HeLa cells. Some properties of the enzyme and of the reaction catalyzed by it have been studied in order to comple- ment previous characterization carried out with crude HeLa cell extracts or partially purified protein [5, 20, 211. The M , determination data indicate that the cyclase is a single poly- peptide protein of 38 - 40 kDa. The anomalous behaviour of the cyclase during Sephacryl gel filtration at low salt concen- tration [20] (and this work) is most readily explained by the ionic interaction of the enzyme with the gel matrix. Sephacryl gel filtration performed in the presence of 0.5 M NaCl led to an M , of 38000, in agreement with M , determinations by other methods.

In the presence of ATP (or other nucleoside triphosphates, as demonstrated in this work) and Mg2+, the enzymecatalyzes the conversion of the 3’-phosphate group to a cyclic 2‘,3’- phosphodiester at the end of RNA substrates. It has been previously proposed [20, 211 that cyclization of 3’-terminal phosphate, catalyzed by the cylase, occurs in three steps:

(a)Enzyme + ATP + Enzyme-AMP + PPi (b)RNA-N3’p + enzyme-AMP + RNA-N3’ppS’A + enzyme (c) RNA-N3’ppS’A + RNA-N > p + AMP.

It is likely that only steps (a) and (b) require the cyclase. Reaction (c) probably occurs non-enzymatically as the result of a nucleophilic attack by the adjacent 2’-hydroxyl on the phosphorus in the diester linkage, as established similarly for the reactions catalyzed by T4 RNA ligase at 3’-phosphory- lated RNA ends, in the absence of 5’-phosphorylated RNA, which is its natural substrate [34]. The covalent labelling of the cyclase polypeptide with [E-~~PIATP (step a) was previously shown by us and by others using partially purified protein [20, 211 and has been confirmed in work described in this paper with the purified enzyme.

Information about step (b) of the reaction was previously provided by experiments carried out with 3’-phosphorylated RNA substrates containing terminal 2’-deoxyribose or 2’-0- methylribose [20]. Incubation of these substrates with cyclase and ATP resulted in the formation of activated 3’-terminal

structures corresponding to dN3’ppS’A or Nm3’ppS’A. When the substrates containing blocked terminal 2’-OH were incu- bated with the partially purified cyclase and CTP or UTP in place of ATP, the cytidylated our uridylated products (con- taining, for example, dN3’ppS’C or dN3’ppS’U structures at the 3’ end) were formed [20]. These results suggested that the cyclase may also use nucleotides other than ATP as cofactors, contrary to the observations of Reinberg et al. [21] indicating that ATP is the only active nucleotide.

Availability of the purified enzyme allowed us to examine directly the nucleotide requirements of the reactions catalyzed by the cyclase. It could be demonstrated that GTP, CTP and UTP can function as cofactors during phosphate cyclization, although much less efficiently than ATP. In agreement with these findings, the formation of covalent complexes of the cyclase with GMP, CMP and UMP has been demonstrated. The acid lability of these complexes suggests that, as in the case of the cyclase-AMP intermediate [21], the other nucleotides are linked to the &-amino group of a lysine residue via a phosphoamide bond.

Other enzymes, such as prokaryotic and eukaryotic DNA (reviewed in [35, 361) and RNA [9, 12, 371 ligases, form covalent adenylylated intermediates from which AMP is transferred to the 5’-phosphorylated DNA or RNA ends; however, modification of these enzymes with nucleotides other than AMP has not been demonstrated. The RNA 3’- terminal phosphate cyclase represents therefore an unusual example of a protein which can form covalent intermediates with several different ribonucleoside 5’-phosphates. It is not known whether this property of the cyclase has any physio- logical significance. Since the K,,, value for ATP is much lower than for GTP (and presumably for the other ribonucleoside triphosphates) it is possible that ATP is the only natural cofactor of the enzyme.

Although all available evidence suggests that cyclization of the RNA 3’-terminal phosphate proceeds via the three steps outlined above, the transfer of AMP from the preformed cyclase-AMP complex to the RNA acceptor has not been directly demonstrated. Reinberg et al. [21], using Sephadex gel filtration, were unable to isolate the active cyclase-AMP complex free of unreacted ATP; it appeared that the complex is unstable in the absence of SDS. Their observation that the most purified cyclase fraction still contained an ATPase activity which hydrolyzed ATP to AMP and PPi was in line with this conclusion; it was conceivable that in the absence of the 3’-phosphorylated RNA substrate the cyclase-AMP complex was rapidly hydrolyzed, resulting in accumulation of AMP and PPi.

In this context it is interesting to note that the preparation of the cyclase described in this work does not contain measur- able activity hydrolyzing ATP to AMP and PPi in the absence of the RNA substrate. This finding suggested to us that the cyclase-AMP complex should be rather stable. Indeed, we have been able to isolated the [32P]AMP-~y~la~e complex free of unreacted ATP by Sephadex gel filtration under nondenaturing conditions, in the presence of 0.5 M NaCl (unpublished results). We are presently attempting to demon- strate directly the function of this complex as an intermediate in the cyclization reaction.

We thank Heide Luther for the protein determination by the phthalaldehyde fluorescent method. We are also indebted to Barbara Hohn, Jan Hofsteenge, Josef Jiricny and Gregory Goodall for critical reading of the manuscript, to Karin Wiebauer for helpful discussions and to Vera Knesel for typing the manuscript. 0. V. was supported by a fellowship from Fundacidn Juan March, Spain.

439

REFERENCES 1. Uchida, T. & Egami, F. (1971) in The enzymes, 3rd edn (Boyer,

P. D., ed.) vol. 4, pp. 205 - 250, Academic Press, New York. 2. Richards, F. M. & Wyckoff, H. W. (1971) in The enzymes, 3rd

edn (Boyer, P. D., ed.) vol. 4, pp. 647 - 806, Academic Press, New York.

3. Konarska, M., Filipowicz, W., Domdey, H. &Gross, H. J. (1981) Nature (Lond.) 293, 112-116.

4. Konarska, M., Filipowicz, W. & Gross, H. J. (1982) Proc. Nut1 Acad. Sci. USA 79, 1474- 1478.

5. Filipowicz, W., Konarska, M.. Gross, H. J. & Shatkin, A. J. (1983) Nucleic Acids Res. 11, 1405-1418.

6. Gegenheimer, P., Gabius, H.-J., Peebles, C. L. & Abelson, J. (1983) J . Biol. Chem. 258, 8365-8373.

7. Schwarti, R. C., Greer, C. L., Gegenheimer, P. & Abelson, J. (1983) J . Biol. Chem. 258,8374-8383.

8. Furneaux, H., Pick, L. & Hurwitz, J. (1983) Proc. Natl Acad. Sci.

9. Greer, C . L., Peebles, C. L., Gegenheimer, P. & Abelson, J. (1983)

10. Phizicky, E. F., Schwartz, R. C. & Abelson, J. (1986) J . Biol.

11. Tyc, K., Kikuchi, Y .. Konarska, M., Filipowicz, W. & Gross, H.

12. Pick, L., Furneaux, H. & Hurwitz, J. (1986) J . Biol. Chem. 261,

13. Perkins, K. K., Furneaux, H. & Hurwitz, J. (1985) Proc. Natl

14. Filipowicz, W. & Shatkin, A. J. (1983) Cell32, 547-557. 15. Laski, F. A,, Fire, A. Z., RajBhandary, U. L. & Sharp, P. A.

16. Stange, N. & Beier, H. (1987) EMBO J . 6, 2811 -2818. 17. Branch, A. D., Robertson, H. D., Greer, C. L., Gegenheimer, P.,

Peebles, C. L. & Abelson, J. (1982) Science (Wash. DC) 217, 1 147 - 1149.

USA 80, 3933 - 3937.

Cell 32, 537 - 546.

Chem. 261,2978-2986.

J. (1983) EMBO J . 2, 605-610.

6694 - 6704.

Acad. Sci. USA 82,684-688.

(1983) J . Biol. Chem. 258, 11 974- 11 980.

18. Kikuchi, Y., Tyc, F., Filipowicz, W., Sanger, H. L. & Gross, H.

19. Kiberstis, P. A., Haseloff, J. & Zimmern, D. (1985) EMBO J . 4,

20. Filipowicz, W., Strugala, K., Konarska, M. & Shatkin, A. J.

21. Reinberg, D., Arenas, J. & Hurwitz, J. (1985) J . Biol. Chem. 260,

22. Peebles, C. L., Gegenheimer, P. & Abelson, J. (1983) Cell 32,

23. Attardi, D. G., Margarit, I. & Tocchini-Valentini, G. P. (1985)

24. Weil, P. A., Segall, J., Harris, B., Ng, S.-Y. & Roeder, R. G.

25. Konarska, M., Filipowicz, W., Domdey, H. &Gross, H. J. (1981)

26. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685. 27. Hashimoto, F., Horigome, T., Kanbayashi, M., Yoshida, K . &

28. Biirk, R. R., Eschenbruch, M., Leuthard, P. & Steck, G. (1983)

29. Bradford, M. M. (1976) Anal. Biochem. 72,248-254. 30. Sista, H. S. (1986) J . Chromatogr. 359, 231 -240. 31. Ackers, G. K. (1964) Biochemistry 3, 723 -730. 32. Fernley, H. N. (1971) in The enzymes, 3rd edn (Boyer, P. D., ed.)

vol. 4, pp. 417-447, Academic Press, New York. 33. Kochetkov, N. K. & Budovskii, E. I. (1972) Organic chemistry of

nucleic acids, part B, Plenum Press, London, New York. 34. Hinton, D. M., Brennan, C. A. & Gumport, R. I. (1982) Nucleic

Acids Res. 10, 1877-1895. 35. Soderhall, S. & Lindahl, T. (1976) FEBS Lett. 67, 1-8. 36. Higgins, N. P. & Cozzarelli, N. C. (1979) Methods Enzymol. 68,

37. Uhlenbeck, 0. C. & Gumport, R. I. (1982) in The enzymes, 3rd edn (Boyer, P. D., ed.) vol. 15, pp. 31 -58, Academic Press, New York.

J . (1982) Nucleic Acids Res. 10, 7521 -7529.

817-827.

(1985) Proc. Nut1 Acad. Sci. USA 82, 1316- 1320.

6088 - 6097.

525 - 536.

EMBO J . 4, 3289- 3297.

(1979) J . Biol. Chem. 254, 6163-6173.

Eur. J . Biochem. 114,223 -227.

Sugano, H. (1983) Anal. Biochem. 129,192-199.

Methods Enzymol. 91, 247 - 254.

50-71.