J. Mol. Biol. (1979) 128, 165-177

Restricted Fragmentation ~AKIO

NoiwoTot,

of Poliovirus Type 1,2 and 3 RNAs by Ribonuclease III

~YUAN

FON

2J~~~

J. DUNN

MANN JACOBSON,

LEES,

~ALEXANDER

AND

~ECKARD

BABICH$ WIMMEH~

‘Department of Microbiology, School of Basic Health Sciences State University of New York at Stony Brook Stony Brook, N.Y. 11794, U.S.A. and 2Department

of Biology, Brookhaven National Upton, N.Y. 11973, U.S.A.

Laboratory

(Received 7 August 1971) Cleavage of the genome RNAs of poliovirus type 1, 2 and 3 with the ribonuclease III of Es&e&&a coli has been investigated with the following results : (1) at or above physiological salt concentration, the RNAs are completely resistant to the action of the enzyme, an observation suggesting that the RNAs lack “primary cleavage sites”: (2) lowering the salt concentration to O-1 M or below allows RNase III to cleave the RNAs at “secondary sites”. Both large and small fragments can be obtained in a reproducible manner depending on salt conditions chosen for cleavage. Fingerprints of three large fragments of poliovirus type 2 RNA show that they originate from unique segments and represent most if not all sequences of the genome. Based upon binding to poly(U) filters of poly(A)linked fragments, a physical map of the large fragments of poliovirus type 2 RNA was constructed. The data suggest that RNase III cleavage of single-stranded RNA provides a useful method to fragment the RNA for further studies.

1. Introduction The enzyme ribonuclease III of Escherichia coli is an endoribonuclease that is able to degrade double-stranded RNA into acid-soluble fragments (Robertson et al., 1968). In vivo, RNase III is known to function as a processing enzyme which cleaves the primary transcripts from the ribosomal cistrons of E. coli and from the early region of bacteriophage T7 at specific sites (Dunn t Studier, 1973a,b; Robertson et al., 1977; Rosenberg and Kramer, 1977). Purified RNase III efficiently cuts unprocessed T7 early RNA in vitro at the same five sites cut in vivo (primary sites) ; however, under certain incubation conditions it cuts less efficiently at additional (secondary) sites (Dunn, 1976). These sites of secondary cleavage also seem to be specific and, in the t Present University, 3 Present 3 Present U.S.A. 7 Author

address: Department of Public Health, School of Pharmaceutical Sciences, Kitasato 6-Q-l Shirokane, Minato-ku, Tokyo, Japan. address: Life Science Department, Bishop College, Dallas, Texas 75241, U.S.A. address: Department of Biology, The Rockefeller University, New York, N.Y. 10021, to whom correspondence

should be addressed. 165

0022%2%36/79/060105-18

$02.00/O

0 1QiQ Academic

Press Inc. (London)

Ltd.

POLIOVIRUS

RNA

FRAGMENTATION

167

RNA fragments for fingerprints were recovered from gels as follows: a piece of gel containing the [32P]RNA was cut out of the slab gel, placed into a lo-ml syringe, mixed 0.02 M-Tris*HCl (pH 6*8), 0.001 M-EDTA, 1% with 1 ml of gel elution buffer (0.5 M-Nacl, sodium dodecyl sulfate) and forced through an Is-gauge needle into a centrifuge tube. The syringe was rinsed twice with 1 ml of gel elution buffer and the mixture agitated at room temperature overnight using a rotator. The gel was pelleted at 10,000 revs/min for 1 h in the Sorvall centrifuge and washed once with 2 ml of gel elution buffer. Both supernatants were combined, mixed with 5 pg of tRNA and the RNAs precipitated with 2 vol. ethanol. The precipitate was dissolved in 0.1 ml of 10 mM-Tris*HCl (pH 7.5), 1 mM-EDTA; the solution heated to 100°C for 2 min, cooled, made O-1 M in NaCl and sedimented through a linear 15% to 30% sucrose gradient, containing 0.1 M-NaCl, 0.01 M-Tris.HCl (pH 7..5), 0.001 M-EDTA and 0.5% sodium dodecyl sulfate. Centrifugation was at 22°C in a Spinco rotor SW41 for 5 h at 41,000 revs/min. The gradient was fractionated; [32P]RNA in peak fractions was precipitated with ethanol, dissolved in buffer and digested to completion with RNase T, (Lee & Wimmer, 1976). RNase T,-resistant oligonucleotides were separated by 2-dimensional gel electrophoresis as previously described (Lee & Wimmer, 1976). (d) Binding

of fragments to poly( U) -jilters

[32P]RNA was incubated with RNase III in the presence of 0.1 M-NH&I as described above. The solution was subsequently mixed with 25 ~1 (1 vol.) of 98% formamide, heated to 60°C for 2 min, chilled to 0°C and finally mixed with 1 ml of ice-cold binding buffer (0.3 iw-NaCl, 0.1 M-Tris.HCl (pH 7.5), 0.1 mM-EDTA). The solution was percolated through a poly(U)-glass fiber filter (Sheldon et al., 1972) at 4°C. The filter was washed 3 times with 10 ml of ice-cold water and 3 times with 3 ml of ice-cold binding buffer prior to application of the sample to the filter. Bound material was eluted 3 times with 1 ml of filter elution buffer (0.01 M-Tris*HCl (pH 7.5), 0.01 M-EDTA, 50% formamide, 0.1% sodium dodecyl sulfate, 5 rg poly(A)/ml). The first elution was at 60°C; the 2nd and 3rd were at 37°C. The combined eluates were mixed with tRNA to a final concentration of 10 pg/ml, with NaCl to 0.2 M and with 2 vol. ethanol. The RNA precipitate was subsequently dissolved in 50 ~1 of gel loading buffer, heated to 60°C for 2 min and electrophoresed in a 2% polyacrylamide slab gel.

3. Results (a) Cleavage of poliovirus

type 1 RNA

with RNase III

At high substrate to enzyme ratios, RNase III cuts unprocessed T7 early RNA predominantly at the five primary sites where cleavage occurs in vivo, provided the concentration of monovalent salt is greater than 0.1 M. Below O-1 M of monovalent sa.lt concentration, primary sites are still the preferred sites of cleavage but now cutting of secondary sites is more readily observed (Dunn, 1976). In order to determine whether poliovirus RNA contains the equivalent of primary or secondary RNase III cleavage sites, we incubated 32P-labeled PVl RNA with a constant amount of RNase III at different concentrations of NH,Cl and then analyzed the products by electrophoresis on polyacrylamide gels (Fig. 1). The amount of RNase III used was known to be in excess over the amount needed to cleave all primary sites in an equal molar amount of unprocessed T7 early RNA. As shown in Figure 1, PVI RNA apparently lacks primary sites, since it is not cut by RNase III when the NH,Cl concentration of the incubation mixture is 0.12 M or greater. Similar patterns were obtained using NaCl or KC1 in place of NH&l. Although poliovirus RNA lacks primary cleavage sites, it does contain numerous secondary cleavage sites, since it is readily cut by RNase III when the NH,CI concentration is reduced below 0.12 M. At constant magnesium (5 mM) and enzyme concentration the cleavage products progressively decrease in size with decreasing NH&l

.\.

No~lo’l’c)

/:“/’

[NH4Cl]

I/..

(u)

POLIOVIRUS

RNA

FRAGMENTATION

169

concentration. At NH,Cl concentrations between 3 mM and 18 mM the cleavage produets become small and cannot be resolved on a 2% gel. As shown in Figure 1 (b) these small fragments are not random cleavage products because they can be resolved into discrete bands when electrophoresed on a 3% to 20% gradient gel. At 78 mM-NH,Cl, RNase III generates predominantly high molecular weight fragments, suggesting that at this salt concentration cleavage occurs at a limited number of preferred secondary sites. Cleavage of the RNA was inhibited in all cases if 5 pg of Penicillium chrysogenum double-stranded RNA, which is a competitive inhibitor of RNase III, were added to the incubation mixture. The presence of 5 pg of E. coli 23 S rRNA did not inhibit the reaction. These observations, together with the failure of RNase III to cut PVl RNA at monovalent salt concentrations greater than 0.12 M, suggest that the RNase III preparation is free of nucleotidohydrolases, such as RNase I. (b) Comparison

of fragmentation

products of polio type 1, 2 and 3 RNAs

Virion RNAs of PVl, PV2 and PV3 have been shown by hybridization to have sequence homology between 30 and 50% (Young et al., 1968). Fingerprints of RNase T,-resistant oligonucleotides of these three viral RNAs, on the other hand, show distinct patterns in the region of large oligonucleotides (Lee & Wimmer, 1976; Lee et al., 1979). We were interested in whether or not these genomes yield similar RNase ITI cleavage patterns, especially under conditions promoting cleavage at a limited number of secondary sites. As can be seen in Figure 2, the cleavage patterns of PVl, PV2 or PV3 RNA at 0*05 and 0.1 M-NH,Cl are distinct when analyzed on 2% gels. All three RNAs have in common, however, their resistance to RNase III at higher salt concentrations and increased susceptibility to the enzyme as the ionic strength in the incubation mixture decreases. The molecular weights of the cleavage products of PV2 RNA generated in O-1 MNH&l have been estimated by gel electrophoresis (Fig. 3) using T7 early mRNAs as standards (Dunn & Studier, 1973a). The sum of the molecular weights of bands I to IV of PV2 RNA exceeds that of the virion RNA by approximately I.8 x 106. The molecular weights of bands I, II and III of PV3 RNA (see Fig. 2) were determined in a similar fashion and found to be: 2.1 x 106, 1.75 x 10s and 0.54 x lo’, respectively (data not shown). The sum of the molecular weights of the PV3 fragments also exceeds that of virion RNA by approximately 1.8 x 106. This observation suggests that some of the fragments must be overlapping. Additional bands to those indicated by roman numerals can be occasionally observed after RNase III cleavage, as for example the weaker bands between I and II, and between II and III of PV2 RNA (Fig. 3). We assume that these bands are less stable intermediates of the cleavage. (c) Mapping

of the RNase III

cleavage products

In order to map the RNase III cleavage sites in PV2 RNA we took advantage of the fact, that all poliovirus RNAs are 3’-polyadenylated (Yogo & Wimmer, 1972; Lee et al., 1979). Therefore, any poly(A)-linked cleavage product must be derived from the original 3’ terminus of the viral RNA. To determine which products were polyadenylated, RNA cleaved in O-1 M-NH&I was filtered through poly(U)-discs (Sheldon et al,, 1972) under conditions that would selectively bind any polyadenylated fragments. The bound material was then eluted and analyzed by polyacrylamide gel

0

0

6 sg

PVI

0

RNA y0

CA R ”

0

6s

PV2

RNA 60 $0

6R ”

0

6z

PV3

RNA 0$

0g z

II

I

[Nti4Ci]

(u)

.-

:. .

Fra. 3. Determination of molecular by polyaorylamide gel electrophoresis Rand 0: undigested 36 S RNA.

Band ISZ

Band III

Band II

Band I

Band 0

.

^

: ,f 0

I

2

3 Distance

4 (cm)

5

Band l!Z (0.48

IO61

6

x 106)

r

weights of the large RNase III-generated fragments of PV2 RNA. Cleavage products of the viral RNA were separated on a 2% slab gel and the distance of migration compared to that of RNase III cleavage products of T7 early mRNA.

.

I-3

0.7

I

Band III (0-84x

Band II (l-24 x IO61

Band I (I.8 x IO61

Band 0 (2.6 x IO61

PV2

BAND

0

BAND

I

BANDII

BAND

III

POLIOVIRUS

RNA

FRAGMENTATION

173

Fro. 6. Fingerprints of RNase T,-resistant oligonucleotides of viral RNA and RNa ,se IIIgenerated fragments by 2-dimensional polymrylamide gel electrophoresis. (a) PV2 [sap ‘]RNA ; (b), (c) and (d) bands II, III and IV, respectively, of an RNase III digest of PV2 [3ZP]RN ‘A. For details, see Materials and Methods.

174

fix~II1

.\.

band

I.

It’so,

hands

I I.

I I

NOMO'I'O

I

anti

E

I \-

mllst

7'

.I

1.

Jx*~Jwstwt

mliqw

portiolis

ot’

tJlr

viral

MNA. To prove this conclusion tve have elut.etl f’ragment.s I I, I LI anti l f’ from the sial) gel and further purified bhe RNA by crntrifugation through I Fi”,, to 300,, suoroht’ gradients. Each fra~gmentj sedimented as it distinct peak (data not ~l1ow11). kak fractions n’ere combined and the RNA digested to c*ompletion with RXase ‘I’, RN:tstb ‘I’,-resistant, oligonucleotides were then sep:~rat(~(l by t\lo-t~imcnsio~~al gt4 elt~t~tr~c,phoresis as previously described (Lee & Wimmw. I Wfi) and t’he fingerprints (v,mpatwi with that of full-length I’\‘:! [32P]RNAA. A h can hta stwi in Figure 5(b), (c) arid (cl). tilt. tingerprint,s of fragments Ll. II I and I\’ diffw frotn t’ach other. ,A ttareful t~otnparisor~ of 25 large oligonucleot~ides 1(Np), , 141 at thcb lo\\w portion of tlw tingerpr’ints iti Figure 5( b). ((2) and (d) suggests that their con1 ~lination nx~~Iltl yield thus pattcwj of Iar.gt~ oli~onucleotidt~s of thtl fingerprint of full-ltsngl Ir Pi’:! RNA (Fig. 5(a)). Sinc.c* it 1t;ld 1,t*en previously sllo1vn that t,llf, large Kl\ast~ ‘I’, -l~t’~iSitil.llt c~ligonut+c~t itIt (t*.g. spoth I t,c, 5) of Plr:! RNA OCCIII~ in unimolw amorrllts in the viral genonw (l,tv cutr/l.. 19%). t htbir sf:p;rratioil among fragment, S I I. I I I tlllti I \’ ht1.tJIlgl?. Sllggt’StS tllt1t tllt’St* fl’ilglllfbnts are distint4 RSA speck &II derived fro1tl I’V:! KKA. In xltiitioii. tlltb sum ot t ht> molrculai~ \2.rights of fragIne11ts I I. I I I atltl 1\. (2.5fi IO”) il~~f?t'S tluitc* 11(bll \vith ill, c,lJst~r~t hc known molecular weight of Pk.2 virion RN=\ (2.7 101’: IAY 4 t/l.. IN!)). vabion indicating that thtwb fragments reprwent rtlc,st. if riot all. stvlut’ntw of’ t lit, P\‘r’ penome. As discussed alJovc>. fragment. < I and I II of I’\.? RN=\ stvtn to IJtb itlitial t-lt~~\-;~g(~ I)roducts (see Fig. 2). Sinw fragments I and I I art’ IJol~ildt~n?.l;ltt,~l. ;I ph>Gn I I11al~ of’ as shtJ\vn in ICgurt~ fi t 1~~~ t~ltwva.pe products of I’\‘2 RNA can tJtb t~~nst rut*ttd

PV2

Poiy(A) 0

m

4. Discussion

A

POLIOVIRUS

RNA

FRAGMENTATION

175

no cutting of viral RNA is observed, although this condition is optimal for processing of primary sites on T7 early mRNA. Thus, the structure of poliovirus RNAs appears to lack primary processing sites. Reducing the ionic strength of the incubation mixture broadens the specificity of cleavage by RNase III such that additional cutting sites (secondary sites) on the RNA are recognized (Dunn, 1976). Both large and small fragments can be obtained, depending on the salt conditions chosen for cleavage, showing that the optimal ionic conditions vary for different secondary cleavage sites on the RNAs. Some care must be exercised in choosing incubation conditions for the preparation of high molecular weight fragments. With PV2 and PV3, cutting occurs at a limited number of sites at monovalent salt concentrations above 50 mM. In contrast, more secondary cleavage sites on PVl RNAs have similar ionic optima. Consequently, the production of clean high molecular weight fragments with this RNA is more difficult. The production of defined fragments of varying length from poliovirus RNAs by RNase III cleavage provides an alternate method to RNase T, or KOH digestion for use in sequence studies. Recently, we have isolated a 5’-terminal RNA fragment of approximately 90 nucleotides using this procedure (Harris et al., 1978). This fragment migrates as a unique band in slab gels regardless of whether it still has a protein linked at its 5’ end (Lee et al., 1977 ; Nomoto et al., 1977) or whether the 5’-terminal protein has been degraded by proteolysis (Harris et al., 1978). Whereas this fragment may have a unique 3’ terminus, it is not yet clear whether cleavage with RNase III under the conditions described here always leads to the production of fragments with unique 5, and 3’ ends as required for utilization with the rapid sequencing techniques (Donis-Keller et al., 1977; Simoncsits et al., 1977). It is to be expected that the methods described here for polio RNA will also be applicable to other large RNA molecules. Adenovirus mRNA has also been cleaved by RNase III when a high enzyme to substrate ratio at 0.15 M-Salt was used (Westphal & Crouch, 1975). Although in these experiments the cleavage products could not be resolved into discrete bands by polyacrylamide gel electrophoresis, some evidence was obtained which suggests specific cleavage. The availability of unique large fragments of poliovirus RNAs is also useful in mapping studies of the poliovirus genome. We have used the fragments in conjunction with oligonucleotide fingerprints to map the location of deletions occurring in DI particles (see the accompanying paper). The cleavage maps of the Iarge fragments can also be used in assessing homogeneity of individual viral stocks. The mechanism of cleavage of single-stranded RNA by RNase III is unknown but seems to depend on a variety of factors that may influence both the properties of the enzyme and the structure of the RNA. The interactions between enzyme and primary sites is very specific at physiological salt concentrations. Sequence studies on the primary cutting sites of T7 early RNA have shown that all of these sites contain basepaired regions and many of the sites contain specific base sequences as well (Robertson et al., 1977; Rosenberg & Kramer, 1977; Young & Steitz, 1978; Dunn, unpublished results). At the lower salt concentration, the enzyme recognizes many more cleavage sites. Possibly this broadened specificity is due to a direct alteration in the conformation of the enzyme. However, the conformation of single-stranded RNA is also salt dependent. Thus, it is to be expected that the conformation of the substrate may also be altered as the ionic strength in the reaction mixture is decreased. The sequence of polio RNAs at the cleavage sites has not yet been determined. Recently, however,

li6

A. ~OlMOTO

fi 7’ .-I 1,.

electron microscopic studies on the secondary structure of poliovirus RLUAs have revealed t,hak large. stable loop structures art 1 associated with at, least two of the cutting sites t,hat lead to tltc production of latyy fragments of PV1 RKA (Jacobson & Wimmcr. unpublished wsults). Although t,licw results art: still prelitninary, it, ib possible t’hat) t’hth prcsw~:~~ of these loop structnt .t‘s wit’hin the RNA may aid in enzyttt( rt:cogtiition of some spetaitk cleavage siks. Besides it,s function >IS a prowssing twzyttrt~ of Art&~-stranded KSA. RSase 1It effectively clra~rs dottl)le-st,t,atlded RNA at. it u itIt> rwrtgc of halt collc:c~ntt,at,ir)tls yield1.5 basr-pairs long (Robertson & J)unn. 1!475). Suring fragments approxitttatt~l~. prisingly, poliovirus ItXX is stable to RN;ast, I I I at ~otict~titt~atiutts Itiplic~r than 0.12 M-NH,Cl. This suggests that dou~)lc-sttwttttt’d region> sufficient for RKasc 111 cleavage may not be accessilk to the action oft ht. c~nzyntc under thest: salt wnditiotts. MternatIiwl~. poliovirus RNAI. \rhiclt is approximately 7800 nttt:lwt~ides long (1,w C$al., 19i9). may not, wntnin pwfwt. hrliwa of I.5 or mow ttnintrtwpt~~tl l,;tw-pairs. This second ittterpret,;ltiott is supporkd tty tw~t~ttt cwtttputt~r studies (III t It