5′-Terminal phosphorylation and secondary structure of double-stranded RNA from a fungal virus

J. Mol. Bid. (1975) 92, 79437

S-terminal Phosphorylation and Secondary Structure of Double-stranded RNA from a Fungal Virus MARIA SZEKELY AND THERESE LOVINY Department

of Biochemistry, Imperial College of Science and Technology London SW 7 2A Y, England

(Received 18 July 1974, and in revised form 13 December 1974) Different double-stranded RNA species from Penicillium etoloniferum virus have been phosphorylated at the 5’ termini with the aid of polynucleotide kinase. A very low phosphate uptake has been observed which, especially in the case of a relatively small moleoular component, was increased several times by pretreatment with RNAase TI. Adenosine and uridine have been detected at the 6’4ermini of this RNA component. Digestion with RNAase Ti, an enzyme which does not cut across the two strands of a double-stranded RNA molecule, produced a new uridine terminus and increased the efficiency of phosphorylation. It is concluded that this double-stranded RNA molecule contains single-stranded stretches at or near the 6’-termini. The possibility of a circular atruoture being formed by the annealing of single-stranded tails is discussed.

1. Introduction A great number of double-stranded RNA viruses have been detected in recent years and this opened up a new field for the investigation of the fine structure of such RNA species. Fungal viruses are a very convenient source of dsRNAi as they can be easily obtained in large quantities and contain several RNA species in a wide range of molecular weights (Wood, 1973). A relatively small molecular component of Penicill&m stolonije~ virus, RNA F-3 (Bozarth et al., 1971), is especially suited for studying the structure of dsRNA, partly because its smaller size makes it accessible to sequencing techniques and partly because it can be isolated in homogeneous form by polyacrylamide gel electrophoresis (Kempson-Jones, 1971). A study on the structure of this RNA has been undertaken earlier (Loviny & Szekely, 1973). In the course of these investigations we also attempted to determine the S/-terminal nucleotides of RNA F-3 by labelling the termini with radioactive phosphate and identifying the labelled nuoleotides after complete hydrolysis. We invariably found pup (or pU, depending on the technique of hydrolysis) as the main component accompanied by a small amount of pA. The results suggested that as opposed to most viral RNAs studied so far, this molecule contains a pyrimidine nucleotide at the 5’-end. The homogeneous RNA F-3 preparation used in these experiments has been, however, carried through a purification step that involved treatment with RNAase T,. Although it had been found that this enzyme does not degrade dsRNA even in low salt concentration or high enzyme: substrate ratio, if single-stranded tails, however short, containing G residues had been present in the RNA molecule. these would have been j-.\bbreviation

unetl: daRNA, double-stranded RX.%. 79

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degraded, thereby producing new 5’-termini. To check whether this was indeed the case, we modified the techniques used for extracting, purifying and separating the different RNA species in P. stoloniferum virus, so as to preserve their native structure and studied the effect of RNAase T, on these molecules. The results, presented here, enabled us to make some suggestions as to the possible secondary structure of RNA F-3 at its 5’-ends.

2. Materials and Methods (a) Virus preparation P. stoloniferum was grown in the Pilot Plant of our Department as described by Banks et al. (1968). Viruses were isolated by the procedure of Banks et al. (1969) with the following modifications. The extract of the mycelium was precipitated by acidification to pH 3.6, without addition of RNA. The precipitate was dissolved in 0.03 M-phosphate buffer (pH 76), debris centrifuged off at 10,000 revs/mm and the virus sedimented by 16-h centrifugation at 17,000 revs/min. The virus preparation was purified by zonal centrifugation in a 10% to 45% sucrose gradient. The fractions containing virus particles were pooled, dialysed against the above buffer and sterilised by Millipore filtering. (b) Isolation of RNA In order to preserve the native structure of the RNAs, a mild extraction procedure recommended by Diener & Schneider (1968) was used. By this technique, RNA is extracted in the presence of sodium dodecyl sulphate and small amounts of phenol which do not form a two-phase system. The virus suspension was incubated at room temperature for 15 min with the phenol reagent, cooled and the RNA precipitated with 3 vol. of ethanol. After several hours at -2O”C, the precipitate was dissolved, the insoluble residue containing denatured protein and salts was discarded, and the solution dialysed against 2% sodium acetate to remove the last traces of sodium dodecyl sulphate. Precipitation of RNA was repeated several times, until no insoluble residue was found and a spectrum characteristic of pure RNA was obtained. The gel electrophoretic pattern of these RNA preparations showed all components in agreement with the data of Bozarth et al. (1971) and of Kempson-Jones (1971). (c) Enzyww and radioadve matwida RNAase T1 was a crystalline preparation of Sankyo Ltd, Tokyo, obtained from Calbiochem. Pancreatic RNAase A, venom phosphodiesterase and alkaline phosphatase from Esciceriohia ~0% were preparations from Worthington Biochemical Corp. [Y-~~P]ATP was obtained from the Radiochemical Centre, Amersham, the specific activity was usually around 16 Ci/mmol when fresh. Polynucleotide kinsee was prepared according to Richardson (1971) from T, phageinfected cells obtained from the New England Enzyme Center, Tufts University, Boston. After the last purification step on an hydroxyapatite column, the active fractions were pooled and pressure-dialysed against a buffer-salt mixture containing 0.025 M-KCl, 0.01 M-mercaptoethanol, 0.02 M-phosphate buffer (pH 7.6). The enzyme was stored in this solution in ice. Enzyme assays were made as described earlier (Szekely & Sanger, 1969). (d) Polyacrytamide gel electrophoreaia Gel slabs, 20 om by 20 cm or 10 cm by 13 cm were prepared and run as described by Adam et a.!. (1969), except that the smaller gels were run horizontally. Bromophenol blue was used as a marker and the run was usually finished when the dye moved about two thirds of the length of the gel. The gel was stained with toluidine blue (0.2% in 0.4 Macetate buffer, pH 4.7) after fixing in 1 N-aCetiC acid for 10 min. The gels were photographed after destaining and exposed immediately for autoradiography. Gels in formamide were prepared according to Staynov et al. (1972). 3.4 g acrylamide, 0.6 g bis-acrylamide, 0.36 g sodium barbitone were dissolved in deionized formamide, the

B/-TERMINAL

PHOSPHORYLATION

OF VIRAL

RNA

x1

pH adjusted to 9.0 with cont. HCI and 0.24 ml N,N,N’,N’-tetramethylethylenediamine and 0.8 ml of a 17 y. (aqueous) ammonium persulphate solution added. The volume was adjusted to 100 ml, the gel poured and left to set in a horizontal template at room temperature. Electrophoresis was carried out in the cold, for 6 to 7 h at 200 V or 16 h at 100 V. Care was taken that the gel should not get into contact with moisture or with aqueous solutions : the filter paper connecting the gel to the electrophoresis buffer was wetted with formsmide and the gel was covered with a thin plastic sheet to protect it from condensation in the covered electrophoresis apparatus. The samples applied to the gel had been dissolved in formamide buffered to pH 9.0 with barbitone and had been denatured by heating at 65°C for 10 to 20 min. The electrophoresis buffer contained 0.02 m-NaCl, 2 g barbitone/l (pH 9.0). RNA was eluted from polyacrylamide gels by a slightly modified technique of Adam et al. (1969). After electrophoretic elution of RNA onto DEAE paper, the papers were washed, dried and incubated for 1 h in a solution of 0.1 mg RNAase/ml in O-0 1 M-Tris *HCl buffer (pH 7.4) in order to fragment the large molecules and thereby improve their recovery from the paper (Szekely et al., 1973). The papers were washed and dried once more and finally eluted with triethylaminecarbonate. Determination of the radioactivity of RNA bands was done by cutting out the bands and counting them in a Beckman 16-200B counter, using Butyl-PBD (Ciba) as scintillation fluid. Different amounts of [Y-~~P]ATP were injected into a similarly sized slice of gel and counted to be used as standards.

(e) 5’-terminal phosphorylation

of RNA

Labelling of the 5’-termini of RNA was carried out by a modified technique of Weiss et al. (1968). In a typical experiment 70 pg RNA were incubated with 12 nmol of [r-3aP]ATP and 5 units of polynucleotide kinase, in the presence of 0.01 M-Tris-HCl (pH 8*0), 0.01 MMgCl,, 0.02 M-mercaptoethanol, 0.01 M-KCl, 0.01 M-phosphate buffer, pH 7.5 (the last two components were contained in the enzyme preparation). The total volume was 33 ~1 and incubation was for 45 min at 37°C. One unit of polynucleotide kinase is defined as the amount that transfers 1 nmol phosphate from ATP to a dinucleoside monophosphate under the conditions of the enzyme assay. If RNAase T, was also included in the reaction mixture, this was present in the proportion of 1: 20 to the RNA if not stated otherwise. After incubation the reaction was stopped by the addition of sodium dodecyl sulphate to 1’5% final concentration and the mixture was either applied directly to polyacrylamide gels (in experiments where the radioactivity of the bands wtas quantitated) or was treated with phenol to re-isolate the RN.4. In experiments when RNA was treated with phosphatase before labelling, this was done according to the two-step prooedure of Szekoly & Sangor (1969). (f) Determination

of the 5’.terrnind

nucleotides

After confirming that the phosphorylated bands detected in the autoradiograph coincided with the bands seen on the stained gel, the band corresponding to the component F-3 (see Plate I) was cut out from the gel and the RNA eluted onto DEAE paper, as described above. The paper was digested with RNAase A and eluted with triethylaminecarbonate. The eluate was incubated with 2 pg venom phosphodiesterase for 2 h at 37’C in 0.05 MTris*HCl (pH 8*9), 0.01 M-MgCI,. The digest was applied to Whatman 3 MM paper and run at pH 3.5 (the electrophoresis buffer contained also 0.001 M-EDTA to prevent streaking) together with mononucleoside mono- and diphosphates as standards. In some cases the eluate was hydrolysed with O-2 x-NaOH instead of by enzymic digestion.

3. Results The viruses grown on P. stolonifewm consist of two species, one contains two dsRNAs of molecular weights I.1 x lo6 and O-94x 108, respectively, the other three dsRNAs of molecular weights 0-99x 106, O-89x lo6 and 0.23~ 106, respectively (Bozarth et al., 1971). The latter is the component RNA F-3. In polyacrylamide gel 6

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electrophoretograms we detected also an even smaller, fast-moving RNA species (band 4 in Plate 1). When using the mild one-phase isolation procedure recommended by Diener & Schneider (1968) to extract the RNAs from the virions, in some preparations we observed traces of an RNA species that did not enter the gel and which probably corresponded to the single-stranded variety of one of the slow bands described by Buck & Kempson-Jones (1973). In other preparations such a band was not detected on the stained gel, but autoradiographs of labelled preparations showed a diffuse slow ba’nd or a diffuse dark background both of which probably were breakdown products of this single-stranded RNA, as they both disappeared upon treatment with RNAase T,. To determine the 5’-terminal nucleotides, the total mixture of the viral RNAs was phosphorylated with polynucleotide kinase and [Y-~~P]ATP and the labelled RNAs were separated by polyacrylamide gel electrophoresis. In this way excessive handling of t#he RNA preparation, which could have destroyed fragile single-stranded parts, was avoided and artifacts, if produced at all by breakdown or side reactions during phosphorylation of the RNA, were separated from the genuine radioactive bands. Plate I shows polyacrylamide gel electrophoretograms of untreated and of phosphorylated viral RNA. As can bc seen on the stained gel (Plate I(a)) treatment with polynucleotide kinase did not cause any changes in the mobility or apparent intensity of the bands. On the autoradiograph of the same gel (Plate I(b), sample B) it is hard bo detect the faintly labelled bands against the diffuse background mentioned above. The position of the radioactive bands corresponds exactly to the stained RNA bands. Their extremely low labelling was an unexpected but perfectly reproducible result. When both the slow bands and the fast one were cut out from a similar gel and their radioactivity determined. the results shown in Table 1 were obtained. Phosphate uptake was very low, a rough estimate based on the known molecular weights and approximate proportions of the different RNA species gives an overall yield of about 1%. In the case of RNA F-3 the phosphate uptake was even lower: less than 1% of the molecules have been phosphorylated. Albhough it is difficult to quantitate a reaction with polynucleotide kinase as it often does not go to completion, such a low efficiency cannot be caused by side reaction or inactivation of the enzyme. It is much more probable t’hat some peculiarity in the structure of the RNA is responsible for t’he low yield. The efficiency of phosphorylation could not be improved by increasing the concentration of polynucleotide kinase (Table 1). H owever, it was increased several times if RNAase T, was added to the phosphorylation mixture as shown in Table 1. It can also be seen in Plate I(b) that in the RNAase-treated sample there is a very substantial increase in the radioactivity of each band. At the same time there is no change in the mobility or in the amount of any of the RNA components. Our kinase preparations always contained traces of phosphatase (Szekely & Sanger, 1969) and therefore it was not usually necessary to dephosphorylate RNA preparations before polynucleotide kinase treatment. (This phosphatase contaminant did not, however, interfere with our obtaining a good yield of phosphorylation when fingerprinting nucleic acids or when assaying the enzyme.) Because of the very low phosphate uptake of these RNAs we checked whether in this case phosphatase pretreatment increased the efficiency of their phosphorylation. As can be seen in Table 1, preparations pretreated with phosphatase showed exactly the same labelling as the untreated samples. This suggested that the 5’-termini of these RNAs might be inaccessible to

A B c

D

E

A

B

PLAg ‘E II. Indentification of 5’.terminal nucleotides in RNA F-3. (a) Paper electroph~ uresis made cdirectly from the RNA digestred as described in Materials and Methods. In eluatcr, from present that caused retardation cIf tho polyacl :ylamide gels there was often some contaminant nueleot ddes when electrophoresed at pH 3.5. The spots marked 1, 2, 3 were therefore eluted from and re-run under identical conditions, using the same standards. Thir this ek ,ctrophoretogram second electrophoresis, shown in (b), was used for proper identification of tho nucleo tides. A, S’-tc ,rmini of untreated RNA; B, 6’-termini of RNA F-3 t,reated wit,h RN&se T,. Tho a1mmws on the left also indicate the position of nucleotide markers.

A

%

C

A

0

I’IATE 111. El~ctrophorrsis of tienaturctl 1’. sfolo~ifer~m virus R&A in formamitle-g&. (a) Stained gel; (b) autoradiograph of the same gel. A. RNA treated with RSAasc T, amI phosphorylated with polynuclt:otide kinasr: B, RNA phoxphorylatctl with polynucleot.idc kinasp. no RNAasc treatment; C, ribosomal RNA standard. f3and 1, undissociat,ed RNL4s S-l, S-2. F-l and F-2: band 2, dissociated RNA. corresponds to half the 31, of HI-C.As S-1, S-2, F-l and F-2: band 3, dissociated RNA, corresponds to half the M, of RNA F-3 : hn.nd 4. small fragmr:nts, 31, about 25,000.

PLATE IV. Production of a small dsRNA fragment from RNA F-3. (a) Autoradiograph of polyacrylamide gel electrophoresis of RN.4 F-Y pret,reatetl with KNAase I’, and phosphorylat,ed with polynucleotide kin&se. A, RNA t,reatod with phosphatase before phosphorylat,ion; B, no phosphatase pretreatment. Band 1, position corresponding to the size of RNd F-3; hantl 2. small fragment,. (b) Electrophoretic identification of the 5’.terminal nucleotitlrs of t,ho bands oluted from gel (a). The eluted RNAs were hydrolysod in 0.2 N-N~OH and run on Whatman 540 paper at pH 3.5 with nuoleoside diphosphate standards. The positions of the lat,tcr arc tihown on t,he right. 1.4 and lR, terminal nnclrntidcn of hand 1 in samplw ;\ a~~1 R. twfwr~ ivltly: L’:\ ~IIII 213. .\ 11111113. ~‘~*spwt i\.t.I>,. itwninal I~wlvotl~lw l)f hnlr~l 2 in tiwnlplw

5’-TERMINAL

PHOSPHORYLATION

OF VIRAL

RNA

83

TABLE 1

32P-u@ake Dy P. stoloniferum virus RNAs Experiment

Conditions

of pho3phorylation

Slow bands

Cts/min in RNA F-3

Total

1

Standard Fourfold concentration of polynuoleotide kinase

12,100 12,380

11,280 12,340

23,380 24,720

2

Standard Pretreatment with phosphatase RNAase T, added

13,800 13,300 51,900

21,400 17,000 127,000

35,200 30,300 178,900

In experiment 1, samples of 10 pg RNA (containing about 0.076 nmol of 6’-termini) were phosphorylated using 1.5 and 6.0 units of polynucleotide kinase, respectively. The whole reaction mixture was applied to a polyaorylamide gel. After electrophoresis the slow bands and band F-3 were cut out and counted as described in Materials and Methods. The counts have been corrected for the decay of saP, Under the same conditions, lo-l2 mol [32P]ATP gave 35,000 cts/min. A rough estimation of the efficiency of phosphorylation is 6% for the slow bands and 0.5% for RNA F-3. In experiment 2 each sample contained 10.8 pg RNA. One sample was pretreated with 0.2 pg phosphatase, the other two samples were carried through the same procedure of adsorption to and elution from phosphocellulose paper (Szekely & Sanger, 1969) without addition of phosphatase. One sample was phosphorylated in the presence of 0.5 pg RNAase T,. Gel electrophoresis and yields of phosphoryla. counting as above. IO-la mol [32P]ATP gave 39000 cts/min. Approximate tion: 5% and 0.7% in the standard mixture for the slow bands and RNA F-3, respectively, 19% and 4%, respectively, in the presence of RNAase T,.

the polynucleotide kinase, not because of the presence of phosphate groups on the termini but possibly because the ends are hidden in some special secondary or tertiary structure. When the 5’-termini of RNA F-3 were determined by eluting the RNA band from the gel and digesting it with pancreatic RNAase and snake venom phosphodiesterase, about equimolar amounts of labelled pup and pA were obtained from the untreated sample. The sample that had been treated with RNAase T, yielded a very great excess of pup (Plate II). The extra phosphate uptake under these conditions seems to be due to phosphorylation of U residues only. RNAase T, thus brings about not only a quantitative hut also a qualitative change in the phosphorylation of 5’-OH groups. RNAase T, is known as a typical “single-stranded” endonuclease. In order to study whether it can break down dsRNA under any special conditions, we investigated its action on the dsRNA of P. chrysogenzcm virus (Edy, Szekely, Loviny & Dreyer, unpublished data). Varying the enzyme:substrate ratio from 1:20 to 1:2, following the reaction for up to two hours, applying different assay techniques, e.g. gel electrophoresis, hyperchromicity a,nd fingerprinting, we did not detect any changes caused by RNAase T, treatment. 14t the same time, complet#e digestion was obtained in 40 minutes at room temperature and in an even shorter time at 37°C with an enzyme: substrate ration of 1:20 if the dsRNA had been denatured before enzyme treatment. We concluded therefore that RNAase T, is not able to cut across double strands. In agreement with these earlier findings, it can be seen in Plate I that no apparent degradation of the dsRNAs occurred upon treatment with RNAase T,.

84

M. SZEKELY

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RNAase ‘I!, however caused some change in the structure of these RNAs, increasing the phosphate uptake in all components and causing a change in the 5’-terminus of at least one of the dsRNAs. In agreement with the specificity of the enzyme discussed above, we must assume that this structural change was brought about by splitting a single RNA strand. There are two ways in which single-strand splits can produce new, accessible 5’-OH groups, without altering the electrophoretic mobility of the RNAs. (i) Nicks may occur in each strand of the dsRNA rather far apart so that the molecule does not fall into pieces. 5’-OH groups at such nicks may not be as accessible to the polynucleotide kinase as terminal groups, still, if they are present in large numbers, they may cause a considerable increase in the total uptake of [32P]phosphate. (ii) Singlestranded region may be present at the end of the molecule which, when split by RNAase T,, produces a new, more accessible 5’-terminus. These two mechanisms can be well differentiated if the RNAase-treated dsRNA is dissociated after phosphoyla. tion: a nicked molecule should yield small fragments, a perfect double-stranded molecule only two single strands each of half the original molecular weight. Experiments comparing intact and RNAase-treated RNA after denaturation showed that both mechanisms were working. Plate III shows electrophoretograms of such RNA preparations, dissociated at 65°C in formamide-containing polyacrylamide gel. Dissociation was not complete under these conditions, some RNA is still detected at a distance which corresponds to its original molecular weight of about 106, the major part of both the slow and fast-moving RNA species run, however, a distance which agrees well with their half molecular weights, 05 x lo6 and O-11x 106, respectively. In this experiment RNA was re-isolated from the reaction mixtures before denaturing it and applying it to the gel. As such treatment may cause some losses, we did not attempt to quantitate the results in this gel. (Quantitation is anyhow less exact when working with denatured RNA than with native molecules, as some of the dissociated strands tend to form aggregates which do not enter the gel.) But even a qualitative comparison of the samples shows two interesting points. On the stained gel it can be seen that the proportion of 100,000 molecular weight RNA strands to 500,000 molecular weight RNA strands has changed upon RNAase T, treatment. The relative amount of 100,000 molecular weight strands decreased, indicating that the enzyme may have caused some random nicking, at least in the RNA F-3 molecule. Correspondingly, a diffuse band appears in the low molecular weight region of the RNAase-treated sample which may represent fragments of this RNA. On the autoradiograph there is a strong spot in the same region. At the same time, however, it is obvious from the autoradiograph that the radioactivity in all the full-size dissociated bands is very strongly increased in the RNAase-treated sample. This is in agreement with mechanism (ii): phosphorylation of a nucleotide at the end of the RNA strands becomes much more efficient. In order to obtain quantitative data on the increased labelling of these nucleotides, the next series of experiments was conducted in a somewhat different way. RNA was phosphorylated in the presence and absence of RNAase T,. Instead of re-isolating the RNA, however, the reaction mixtures were freeze-dried and treated with formamide at 65°C directly, in order to avoid losses which may occur when RNA is re-extracted with phenol. The denatured mixtures were then applied to formamide-containing gels. Some loss of RNA which did not enter the gel could not be prevented, but as the control and RNAase-treated samples were handled in exactly the same way, the

5’.TERMINAL

PHOSPHORYLATION

OF VIRAL

RNA

85

experiments were repeated several times and the gels were stained before autoradiography, it was reasonable to assume that the amount of RNA was comparable in the two samples. The bands corresponding to the undissociated fraction (lo6 M,) aa well as those of the dissociated, 0.5 x lo6 molecular weight strands were cut out and their radioactivity determined. As shown in Table 2, the increased labelling of the RNAasetreated sample appears as well in the dissociated strands as it does in the undissociated molecules. This proves that, at least in the slow-moving RNAs, the extra phosphate uptake occurs at the ends of the strands. Nicked strands, if any, would have been separated from the 500,000 molecular weight band in the formamide gel.

TABLE

2

““P-content of slow RNA bands itb forwmrnide-gel

Treatment

of RNA

Standard + RNAase

T,

Cts/min Undissociated RNA 8,860 20,600

in bands Dissociated RNA 14,400 41,100

In the case of RNA F-3 some nicking occurs and this causes changes in the amount of the RNA present in the 110,000 molecular weight band. Direct counting as above would therefore give ambiguous results. The RNA eluted from these bands was therefore used only for identification of the S’terminal nucleotides. By digestion with pancreatic RNAase A and venom phosphodiesterase, pA and pup were obtained in about equimolar amounts from the dissociated strands of the untreated RNA and practically only pup, with merely traces of pA, were found in the dissociated strands of the RNAase-treated sample. Finding the same terminal nucleotides, whether dsRNA or dissociated strands are investigated, proves that they originate from the ends of the strands and were not produced by random nicking. It can thus be concluded that RNAase T, acts by splitting near the end of the RNA molecules and in this way produces new 5’-termini which are more accessible to phosphorylation than the original ones. Considering the specificity of RNAase T, for single RNA strands, it has to be assumed that there are single-stranded stretches in these molecules at or near their 5’-ends. In the case of RNA F-3 the single-stranded stretch must contain at least one G-U sequence.

4. Discussion The above results allow some speculation about the secondary structure of RNA F-3. An obvious assumption is that the molecule has a single-stranded tail(s) at one or both 5’-ends. These could be split by RNAase T, after G residues, and one or two new termini could be produced. This hypothesis does not agree, however, with all our observations. It does not explain why the original molecule is not readily phosphorylated and it does not account for the appearance of new bands in the polyaorylamide gels upon RNAase T, treatment.

M. SZEKELY

86

AND

T. LOVINY

In Plate I it can be seen that the RNAase-treated samples contain an extra band (band X), a well-defined RNA component. This band disappeared if the RNA preparation was treated with pancreatic RNAase in low-salt media, while it was not attacked by the same RNAase in high-salt media. This indicated that this RNA fragment is double-stranded. A rough estimate of its molecular weight, based only on its mobility in these gels gives a value around 50,000. Its size is too large to be a split product of RNA F-3, as a reduction in the size of the latter should then be detectable, but it may arise from one of the slow-moving RNA species. When a very short electrophoretic run was made with a homogenous RNA F-3 preparation that had been treated with RNAase T, (Loviny C Szekely, 1973) in the course of purification, we detected an even smaller RNA fragment which may have been split off this molecule and which was also quite well phosphorylated (Plate IV(a)). The 5’-termini of this fragment were determined and found to be A and U, i.e. identical to the original 5’-termini of the RNA F-3 molecule, while the RNA band in the proper position for F-3 yielded almost entirely U termini with only a trace of A, as usual (Plate IV(b)). It is possible to construct more than one model for the structure of RNA F-3 which fits most of the data discussed here. A linear double-stranded structure with a short single-stranded stretch not exactly at the end of the molecule, but near to it, would account for the small dsRNA fragments produced by RNAase T,. It would offer no explanation, however, for the low phosphate uptake of the molecule. It can also be considered that if substituted bases were present at the 5’-termini, this could cause poor phosphorylation. It seems improbable though that such bases should never have been detected in RNA F-3 and should not show up in the present electrophoretograms either (see Plate II). Miura et al. (1974) found a substituted base at one of the termini of dsRNA from cytoplasmic polyhedrosis virus and this RNA also showed a low phosphate uptake, but phosphorylation in their experiments could be increased by removing some nucleotides from the complementary strand. It seems therefore that it is more the secondary structure around the 5’-termini and not the nature of the terminal nucleotide itself which determines the extent of phosphorylation. In the following paragraph we propose a hypothetical model for RNA F-3 which is in good agreement with all our findings. Such a structure seems probable also because it is analogous to the structure of some dsDNA molecules. If there are short single-stranded tails at both 5’-ends of the F-3 RNA molecule, and if these contain complementary base sequences, then by the annealing of the tails RNA F-3 u A U U A -A

\ 58 /f %h, \ c3

s

U _ -i

(U)

(U) (U)

FIG. 1. Model for the formation of a circular structure of RNA F-3 and its splitting by RNAase T,. Arrows show the sites where RNAese T, can attack. Letters in parentheses indicate new 6’-tmmini.

6’-TERMINAL

PHOSPHORYLATION

OF VIRAL

RNA

87

a circular structure may be formed (Fig. 1). Two very short single-stranded stretches may still remain in the circular molecule between the 5’- and 3’-ends of each strand.

RNAase T, may split at one or both of these stretches. The original 5’-terminal nucleotides, A and U, are not readily accessible as they are part of the double-stranded structure and may be even more buried in the tertiary structure if the circle is twisted. One split by RNAase T, could expose one of the origina. 5’-termini (also U), while A,

the other original terminus may still not be freely accessible, being part of the doubleskanded structure. Two RNAase splits would produce a small double-stranded fragment containing the original 5’-termini and a linear molecule slightly shorter than the original RNA F-3 which contains two new, fully-exposed 5’-terminal nucleotides,

both U. This agrees with the results of the experiment shown in Plate IV. This work was supported

in part by a grant from the Science Research Council.

Note added in proof: Blocked 5-‘terminal structures have been recently detected in messenger RNAs from dsRNA viruses (Rottman et al., 1975 and Furuichi & Miura, 1974). A methylated G residue is attached in a 5’--5’ pyrophosphate linkage to the B/-terminus. Although such a structure was found only in transcripts of the viral RNA, these results may be relevant to our findings as these molecules are also resistant to phosphorylation with polynucleotide kinase. REFERENCES Adam, J. M., Jeppesen, P. G. N., Sanger, F. & Barrell, B. G. (1969). Nature (London), 223, 1009-1014. Banks, G. T., Buck, K. W., Chain, E. B., Himmelweit, F., Marks, J. E., Tyler, J. M., Hollings, M., Last, F. T. C Stone, 0. M. (1968). Nature (London), 218, 542-545. Banks, G. T., Buck, K. W., Chain, E. B., Darbyshire, J. E. & Himmelweit, F. (1969). Nature (London), 223, 155-158. Bozarth, R. F., Wood, H. A. & Mandelbrot, A. (1971). Virology, 45, 516-523. Buck, K. W. & Kempson-Jones, G. F. (1973). J. Gen. Viral. 18, 223-235. Diener, T. 0. & Schneider, I. R. (1968). Arch. Biochem. Biophys. 124, 401-412. Furuichi, Y. & Miura, K. (1974). Nature (London), 253, 374-375. Kempson-Jones, G. F. (1971). Ph.D. Thesis, Imperial College, London. Loviny, T. & Szekely, M. (1973). Eur. J. Biochem. 35, 87-94. Miura, K., Watanabe, K. & Sugiura, M. (1974). J. Mol. Biol. 86, 31-48. Richardson, C. C. (1971). In Procedures in NucZeic Acid Research (Cantoni, G. L. and Davies, D. R., eds), vol. 2, pp. 815-828, Harper & Row, New York. Rottman, F., Shatkin, A. J. & Perry, R. P. (1975). Cell, 3, 197-199. Staynov, D. Z., Pinder, J. C. & Gratzer, W. B. (1972). Nature New BioZ. 235, 108-110. Szekely, M. & Sanger, F. (1969). J. Mol. BioZ. 43, 607-617. Szekely, M., Brimacombe, R. & Morgan, J. (1973). Eur. J. Biochena. 35, 574-581. Weiss, B., Live, T. R. & Richardson, C. C. (1968). J. BioZ. Chem. 243, 4530-4542. Wood, H. A. (1973). J. Gen. ViroZ. 20, 61-85.