Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 3077-3081, August 1975 Biochemistry
Cleavage of adenovirus messenger RNA and of 28S and 18S ribosomal RNA by RNase III (cell-free protein synthesis/gel electrophoresis of RNA fragments)
HEINER WESTPHAL AND ROBERT J. CROUCH Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014
Communicated by Bernhard Witkop, June 9,1975
Escherichia coli ribonuclease III cleaves adABSTRACT enovirus messenger RNA and mammalian 28S and ISS ribosomal RNA. Fragmentation is not random, but in each case a specific collection of products is generated. This points to the potential use of the enzyme as a tool for specific fragmentation of RNA. Cleavage by RNase III abolishes the capability of adenovirus messenger RNA to direct cell-free synthesis of virus polypeptides.
RNase III, an enzyme found in extracts of Escherichua coli, was originally thought to act exclusively on double-stranded RNA (1) generating oligonucleotides (2-4). Recently, however, considerable attention has been focused on the fact that precursors of both bacterial (5-8) and eukaryotic (9) rRNA, as well as the primary transcripts of early T7 DNA (6, 10, 11), are cleaved by the enzyme into fragments which are indistinguishable from those observed in vivo, implying a role for RNase III in the processing of RNA. At first sight, nearly complete degradation of doublestranded RNA and precise fragmentation of rRNA precursors or T7 transcripts are seemingly unrelated features of one enzyme. Yet, these two properties appear to copurify both genetically (5) and biochemically (4). It has been suggested that hairpin loops within the RNA molecule may be recognized by the enzyme (11, 12), possibly in conjunction with some signals conveyed by the primary structure next to the cleavage site (10, 8). Thus, base-paired regions of RNA may be the common denominators for recognition by RNase III. Helical structures have been most elegantly visualized in precursors of mammalian rRNA (13), and may be common as well to messenger RNA (14). A gradient of susceptibility of RNA sites to RNase III cleavage may be envisaged, with primary sequence and steric conformation determining the various degrees of exposure to enzymatic attack. While the signals for initial processing of large T7 transcripts and rRNA precursors are strong enough to elicit prompt response by the enzyme, more vigorous cleavage conditions may be needed for less exposed sites. Using three RNAs, adenovirus type 2 (Ad2) mRNA, 28S rRNA, and 18S rRNA, we will show here that unique patterns of fragments indeed result from cleavage by high doses of RNase III. MATERIALS AND METHODS RNase III was prepared as described (3). Upon isoelectric focusing of the material, one major band appears (R. J. Crouch, unpublished), which contains the RNase III activity. Ad2 mRNA. RNA was isolated from polysomes of KB cells, pulse-labeled with [3H]uridine at late stages of producAbbreviation: Ad2, adenovirus type 2. 3077
tive infection with Ad2. Poly(A)-containing RNA sequences were selected by oligo(dT)-cellulose chromatography (15). The preparation of [32P]mRNA followed the same protocol, except that cells were starved in phosphate-free medium (containing 2% dialyzed fetal calf serum) for a period of 2 hr preceding the pulse. A 2-hr incubation of 6 X 108 cells in 200 ml of medium, containing 7.5 mCi of carrier-free 32PO4, resulted in the preparation of 0.4 mg of mRNA with an initial specific radioactivity of 105 cpm/,ug. Less than 5% of the radioactivity of the Ad2 mRNA preparations was resistant to RNase A cleavage (for test conditions, see ref. 16). Cell-Free Protein Synthesis. Ad2 mRNA was translated in an S30 system of Krebs II ascites cells (15), with the following modifications. The assays contained up to 15 ng of exogenous mRNA per ll, 3.6 mM magnesium acetate, and 100 mM KC1. Translation products were analyzed by electrophoresis on 13% sodium dodecyl sulfate/polyacrylamide gels, and the radioactivity contained in bands of individual viral polypeptides was determined as reported (15). KB Cell rRNA. Growing cells were washed with, and resuspended in, phosphate-free medium containing 2% dialyzed fetal calf serum and 10 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), added to stabilize the pH (17). After 2 hr, 5 mCi of carrier-free 32pO4 was added to a culture containing 3 X 107 cells in 50 ml of medium, and 24 hr later cytoplasmic RNA (0.2 mg; 6 X 105 cpm/,ug) was prepared (15). Individual species of rRNA were gained by fractionation of cytoplasmic RNA on 1530% neutral sucrose gradients (16). Fractionated RNA was precipitated with ethanol, redissolved in H20 at 70 jig/ml, and stored for several weeks in liquid nitrogen, without noticeable degradation.
RESULTS Electrophoresis of RNA fragments cleaved from Ad2 mRNA and KB cell rRNA The observation of hairpin loops in ribosomal (13) and in Ad2 mRNA (J. Maizel, personal communication) and the suggestion that helical structures within a ribonucleic acid chain convey signals for RNase III attack (11, 12) prompted us to test the susceptibility of mammalian rRNA and of Ad2 mRNA to this enzyme. Based on the known preference of RNase III for doublestranded RNA (1), the enzyme dose was calibrated with the synthetic ribopolymer poly[r(A-U)] (3). An amount of RNase III sufficient to render >99% of the poly[r(A-U)] Cl3CCOOH-soluble did not change the capability of Ad2 mRNA to direct cell-free synthesis of Ad2 polypeptides. However, a further increase of the enzyme dose resulted in
3078
Biochemistry: Westphal and Crouch
Proc. Nat. Acad. Sci. USA 72 (1975) 1
2
3
4
5
6
8
7
4
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100
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FIG. 1. Sucrose gradient analysis of degradation of Ad2 mRNA with RNase III and protection of degradation by duplex RNA. RNA (4.75 Mg) was incubated in 100 Ml containing 0.15 M KCl, 0.01 M MgCl2, 1 mM dithiothreitol, and 0.05 M Tris-HCl, pH 7.9, for 30 min at 37°. After an equal volume of a solution containing 10 M urea (Schwarz/Mann, ultrapure), 50 mM EDTA (neutralized with NaOH), and 2% sodium dodecyl sulfate was added, the reaction mixture was boiled for 1 min. Dilution with 133 MAl of H20 was followed by centrifugation at 4° for 220 min at 55,000 rpm in a 1530% (w/v) sucrose gradient, containing 0.1 M NaCl, 0.1 mM EDTA, 0.01 M Tris.HCl, pH 7.0, in a Spinco SW56 rotor. A parallel gradient contained KB cell [14C]rRNA as a size marker. Fractions were collected and radioactivity of aliquots was determined in scintillation fluid. (a) No enzyme, no duplex RNA; (b) 68 units of RNase III, 200 ,g of duplex RNA, isolated from replicative intermediates of bacteriophage MS2 RNA (a gift from Dr. Jerome Birnbaum, Merck Institute, Rahway, N.J.); and (c) 68 units of RNase III, no duplex RNA (see ref. 1 for definition of units).
the generation of Ad2 mRNA fragments that sedimented bea sucrose gradient (Fig. ic). A concern that it was not RNase III but a contaminating enzyme that was responsible for the observed decrease in sedimentation of the RNA was resolved by two types of experiments. First, addition of double-stranded RNA prevented the cleavage of Ad2 mRNA (Fig. lb). Second, under conditions of large excesses of Ad2 mRNA, complete protection of poly[r(A-U)] from degradation by RNase III was observed (data not shown). These results provided evidence that the sites cleaved in Ad2 mRNA and poly[r(A-U)] were attacked by the same enzyme, RNase III. Encouraged by the susceptibility of Ad2 mRNA to RNase III attack, we exposed mature mammalian ribosomal 28S and 18S RNA to comparable doses of enzyme and analyzed -the cleavage products by polyacrylamide gel electrophoresis. To allow for good resolution of all sizes of RNA between 28S and 4S, the gel was poured in two stages, with a gradient of acrylamide on the bottom and an agarose-acrylamide composite gel on top. The interphase between the two parts of the gel was clearly visible in the autoradiogram (Fig. 2). Ad2 mRNA incubated in the absence of enzyme displayed severtween 18S AND 4S rRNA markers in
.,
_
4S
FIG. 2. Polyacrylamide gel electrophoresis of RNase III-treated Ad2 mRNA and KB cell rRNA. Incubations of RNA and enzyme (see table below) were stopped by the addition of 1 volume (20 Mul) of 0.05 M EDTA, 10 M urea (Schwarz/Mann, ultrapure), 2% (v/v) 2-mercaptoethanol, 0.02% (w/v) bromophenol blue, 0.1 M NaPO4, pH 6.8,0.2% (w/v) sodium dodecyl sulfate, heated 1 min in boiling water, cooled to room temperature, and placed into preformed slots of a 1.5 mm (thickness) X 300 mm (length) X 160 mm (width) slab gel (35). The upper half of the gel contained 2.65% (w/v) acrylamide (Eastman) and 0.5% (w/v) agarose (Sigma), whereas the lower half of the gel consisted of a linear gradient of 3.5-20% acrylamide, stabilized by 0-10% (w/v) sucrose (Schwarz/ Mann, ultrapure). The concentration of N,N'-methylene-bisacrylamide (Eastman) was 0.08% (w/v) throughout the gel. Electrophoresis in 0.05 M NaPO4, pH 6.8, 0.1% sodium dodecyl sulfate, 2 mM EDTA (35) was at 15° for 17 hr at 350 V in a BioRad model 221 slab gel apparatus. The gel was briefly rinsed with water, dried under vacuum, and autoradiographed (35) for 2 days.
Gel slot 1 2 3 4 5 6 7 8
Substrate
Enzyme
RNA
ng
cpm
units
Ad2 mRNA Ad2 mRNA KB polysomal RNA KB 28S rRNA KB 28S rRNA
400 400 100 140 280 140 112 112
40,000 40,000 33,000 46,000 92,000 46,000 37,000 37,000
0 5.1 0 0 0.34 8.5
KB 28S rRNA KB 18SrRNA KB 18S rRNA
8.5 0
1-
Biochemistry: Westphal and Crouch M
b
a
c
d
Proc. Nat. Acad. Sci. USA 72 (1975)
3079
e
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50
FIG. 4. Loss of translation capacity of individual Ad2 mRNAs as a function of RNase III cleavage. The radioactivity contained in the polypeptide II and pVII bands of columns a-e, Fig. 3, was determined (15), and expressed as percent fraction of the control (100% refers to 3020 cpm of polypeptide II and 3855 cpm of polypeptide pVII, column a, Fig. 3).
pVIIVil-
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FIG. 3. Cell-free translation of RNase Ill-treated Ad2 mRNA. RNA (260 ng) was incubated in 10-gl assays with (a) 0, (b) 0.08, (c) 0.19, (d) 0.28, or (e) 0.53 unit of enzyme. No RNA or enzyme was added to a control assay (f). At the end of the incubation period, 5-,ul aliquots of each assay were added to 55 ,l of translation mixture. Translation products obtained in this reaction (% of the reaction mixture) were analyzed by electrophoresis through a 13% sodium dodecyl sulfate/polyacrylamide gel (15) (thickness 1.5 mm, length 300 mm, width 160 mm) at room temperature for 15 hr at 200 V. The figure represents an autoradiogram of the dried gel, depicting 35S-labeled polypeptide bands. Roman numerals refer to individual polypeptide components (23) of an adenovirion preparation (15), added as a size marker (M).
al bands which, although visible in the original autoradiopoorly resolved in the photograph (Fig. 2, column 1). Nevertheless, the shift of RNA radioactivity toward greater electrophoretic mobility after enzyme treatment (Fig. 2, column 2) was obvious. Much more distinct were the patterns of RNA fragments resulting from the treatment of purified 28S rRNA (Fig. 2, column 4) with RNase III. Whereas, a low dose of enzyme produced fragments that migrated in several bands, some between 28S and 18S rRNA markers, one near the bottom of the gel (Fig. 2, column 5), a high amount of enzyme resulted in further fragmentation, as evidenced by a number of bands displayed in the lower half of Fig. 2, column 6. No radioactivity remained at the origin, indicating that all 28S rRNA had been cleaved. On the other hand, 18S rRNA apgram, were
peared more resistant to RNase III attack, with only a portion of the RNA cleaved into fragments which formed a pattern distinct from that of 28S rRNA cleavage products. Translation of RNase Ill-treated Ad2 mRNA A number of virion polypeptides are synthesized in cell-free systems directed by late Ad2 mRNA (15, 18, 19, 16, 20-22). In an attempt to correlate the observed fragmentation of the RNA by RNase III with its biological activity, we translated enzyme-treated Ad2 mRNA in an S30 extract of Krebs II ascites cells. This cell-free system had a considerable background of endogenous protein synthesis in the absence of added mRNA, as demonstrated in column f of Fig. 3, displaying the electrophoretic pattern of polypeptides synthesized in the control assay. Polypeptide migration was toward the bottom of the gel, separating polypeptides in the size range of >105 to about 104 daltons. Addition of 30 ng of Ad2 mRNA to the cell-free system resulted in the synthesis of a number of virion protein components, some of which could easily be discerned (column a, Fig. 3) using adenovirion polypeptides (column M) as a reference. Treatment of Ad2 mRNA with increasing doses of RNase III prior to cellfree translation led to the gradual disappearance of the polypeptides specified by Ad2 mRNA (columns b through e). Yet, the sensitivity of in vitro expression to enzyme attack varied considerably among individual virus-specific polypeptides. For instance, the amount of RNase III required for a 50% reduction in the synthesis of polypeptide pVII, the precursor (23) of core protein VII, was more than twice that needed to reduce hexon (= polypeptide II) synthesis to the same level (Fig. 4). Ad2 mRNAs coding for individual components of the virus particle have been partially separated by sucrose gradient centrifugation (18, 22, 20). The size of some of these mRNAs, estimated from their sedimentation velocities, exceeds that required to specify the respective polypeptide. For instance, pVII, a small polypeptide of 20,000 molecular weight (23), is expressed by an RNA of perhaps as much as five times (18) the pVII code size. This has led to the suggestion that the RNA may be polycistronic, i.e., coding for
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Proc. Nat. Acad. Sci. USA 72 (1975)
more than one polypeptide (18). If so, treatment of Ad2 mRNA with RNase III might generate fragments that retain the coding capacity for an entire virus polypeptide, provided the enzyme does not destroy signals required for proper translation. Accordingly, we treated Ad2 mRNA with increasing amounts of RNase III (corresponding to assays a through e, Fig. 3), fractionated the cleavage products by sucrose gradient centrifugation, and tested the RNA contained in individual gradient fractions in translation assays. The sedimentation profiles of the Ad2 mRNA (Fig. 5) showed a gradual shift of radioactivity from a position between 28S and 18S rRNA (assay a = Ad2 mRNA incubated in the absence of RNase III) to a position close to 4S (assay e = highest enzyme concentration). RNA contained in individual fractions of the sucrose gradient gave rise to the cell-free translation products displayed in the electropherograms at the left of Fig. 5. The virus-specific protein that could be most readily discerned from the background of endogenous material is polypeptide II or hexon, visible as the top band in the columns corresponding to fractions 7-12 of panels a-d. Most of the mRNA giving rise to polypeptide II appeared to be contained in fractions 9-10, in quantities inversely proportional to the amounts of enzyme used. Like polypeptide II, none of the smaller virus-specific polypeptides, such as polypeptides III, IV, or pVII, varied in position in the sucrose gradient, no matter which concentration of RNase III had been used (note that pVII is present, albeit poorly resolved, in panels a and c); moreover, no additional polypeptide bands were detectable among the translation products of RNase III-treated Ad2 mRNA. Thus, we were unable to detect fragments of Ad2 mRNA that retained their ability to specify cell-free translation products.
0 -D
3. 5- 7- 9- 11-13-15-17-
4 6 8 10 12 14 16 18
DISCUSSION Two distinct types of eukaryotic RNA, adenovirus messenger RNA and KB cell ribosomal RNA, were found susceptible to attack by RNase III. With each RNA, unique patterns of fragments were observed. Certain RNA sites were more sensitive to cleavage than others, as evidenced by the appearance of large fragments of 28S rRNA which were further cleaved by a higher dose of enzyme. These findings are compatible with the postulate that RNase III, an enzyme with known specificity for double-stranded RNA, attacks helical structures in each of the RNAs tested, and that a gradient exists of susceptibility of individual RNA sites to enzymatic cleavage. If so, RNase III, as well as endonucleases with comparable properties may assume the role of tools for RNA analysis in a manner analogous to that of restriction enzymes in the analysis of DNA. For instance, the enzyme might become important for the analysis of regions of nonhomology distinguishing closely related RNAs, for RNA sequence determinations, for examining new pathways of RNA processing, and for isolating RNA segments of special interest, such as the variable region within an immunoglobulin RNA chain. RNase III degrades double-stranded RNA into fragments 15-20 nucleotides in length (2-4). Possibly, helical structures within a single RNA chain are degraded in a similar fashion. For instance, an RNA chain containing a single helical region susceptible to enzyme attack would thus be cleaved into two polynucleotide fragments corresponding to the RNA segments on either side of the helix. An oligonucleotide, corresponding to the helical region, would also be produced.
5
10
15
FRACTION NUMBER FIG. 5. Sucrose gradient centrifugation and cell-free translation of Ad2 [3H]mRNA cleaved by RNase III. Aliquots (75 gl) of RNase III assays a-e (Fig. 3) were sedimented through sucrose gradients (see Fig. 1), and the radioactivity of 0.25 volume of each gradient fraction was determined in scintillation fluid. The RNA sedimentation profiles appear at the right side-of the figure. The remainders of the sucrose gradient fractions were combined, as indicated at the left of the abscissa, adjusted to 0.15 M KCl, and precipitated by ethanol, together with 4 ,ug of carrier rabbit tRNA (General Biochemicals). The samples were centrifuged overnight at -20° in a swing-out rotor at 1800 X gmax, washed with 70% (v/v) ethanol in water, recentrifuged for 30 min, dried, and taken up in 60 ,ul of translation mixture. The 35S-labeled products of the cellfree translations were electrophoresed on standard 13% sodium dodecyl sulfate/polyacrylamide gels (15). The autoradiograms of these gels, shown on the left side of the figure, include adenovirion marker proteins (M), aliquots of the translation assays of Fig. 3 (A), and translation assays lacking exogenous mRNA (B). Roman numerals at the left ordinate refer to individual adenovirus poly-
peptides (see Fig. 3).
We have made no attempts to account for fragments of oligonucleotide size and, therefore, have not data to support the mechanism of cleavage envisioned here. In any event, RNase III treatment of Ad2 mRNA did not generate significant amounts of radioactivity remaining on top of the sucrose gradients (Fig. 5). This indicates that the fraction of RNA, if any, that is degraded to oligonucleotide size must be small, or, conversely, that most or all of the RNA is represented in fragments of polynucleotide size. A strong link between the structural integrity of messenger RNA and its biological activity is indicated by the find-
Biochemistry: Westphal and Crouch ing that exposure of Ad2 mRNA to RNase III destroyed its capability of directing cell-free synthesis of virus-specific polypeptides, a result which could explain the inhibitory effect of high concentrations of RNase III on the cell-free translation of encephalomyocarditis RNA (24). Evidence has accumulated which suggests that eukaryotic mRNA contains more information than is necessary to encode for the cognate protein (25-29, 36). The function(s) of such extra RNA is, as yet, unknown. It is conceivable that the rapid inactivation of Ad2 mRNA to act as a message in a cell-free system after treatment with RNase III reflects a preference of the enzyme for sites present in the extra RNA regions and not an attack in the coding sequence. Certainly if RNase III action separates the coding sequence from any signal required for translation, the RNA would be inactivated without the necessity of a cleavage in the region of the encoded message. Recently, Both et al. (30) have proposed that the 5'-terminal 7-methyl guanosine is necessary for efficient translation of vesicular stomatitis virus and reovirus mRNA. If the Ad2 mRNA has a similar requirement, simply removing a few nucleotides from the 5' terminus would render the mRNA inactive for in vitro protein synthesis. The greater the distance the coding sequence is from the 5'-terminal methylated nucleotide, the greater is the chance for separation of the translated sequence and the 5' terminus. Even though the mRNA for pVII sediments as if the RNA is five times (18) the pVII code size, we find the mRNA, after treatment with RNase III, in a translatable form only in the region of the sucrose gradient in which untreated pVII mRNA is found. Such an observation is consistent with either a direct inactivation of the pVII mRNA by nucleolytic action in the coding sequence or by a severing of the translatable sequence from a structure or sequence necessary for proper translation. In light of this consideration, the preservation of translatable Ad2 mRNA sequences after RNase III attack would seem indeed unlikely. The in vitro synthesis of the hexon polypeptide is more affected by RNase III action than that of pVII, the precursor to core polypeptide VII. Hexon mRNA sediments ahead of pVII mRNA in sucrose gradients. This suggests that, as the larger target, it is more vulnerable to RNase III attack. Alternatively, or in addition, the Ad2 mRNA preparation may contain more copies of pVII mRNA than of hexon mRNA, or hexon mRNA may harbor sites more sensitive to RNase III than those of pVII mRNA. The list of RNases of both prokaryotic (31) and eukaryotic (32, 33) origin with limited and specified endonucleolytic action is growing rapidly. Even RNase T1, when tested under restricted conditions (34), may be included in this list. This area of research may be expected to bring deeper insight into the various pathways of RNA processing and degradation and their regulation in vivo while generating more refined methods for RNA analysis in vitro. We thank Dr. J. J. Dunn for helpful discussions, Dr. F. -J. Ferdinand for Ad2 [ssP]mRNA, Mrs. S.-P. Lai for superb technical assistance, Drs. H. D. Robertson, J. J. Dunn, D. Ginsburg, and J. A. Steitz for making their results available to us prior to publication, and Dr. Jerome Birnbaum for his generous gift of duplex RNA.
Proc. Nat. Acad. Sci. USA 72 (1975)
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1. Robertson, H. D., Webster, R. E. & Zinder, N. D. (1968) J. Biol. Chem. 243,82-91. 2. Schweitz, H. & Ebel, J. P. (1971) Biochimie $3, 585-593. 3. Crouch, R. J. (1974) J. Biol. Chem. 249,1314-1316. 4. Robertson, H. D. & Dunn, J. J. (1975) J. Biol. Chem. 250, 3050-3056. 5. Nikolaev, N., Silengo, L. & Schlessinger, D/ (1973) J. Biol. Chem. 248,7967-7969. 6. Dunn, J. J. & Studier, F. W. (1973) Proc. Nat. Acad. Sci. USA 70,3296-3300. 7. Nikolaev, N., Schlessinger, D. & Wellauer, P. (1974) J. Mol. Biol. 86,741-747. 8. Ginsburg, D. & Steitz, J. A. (1975) J. Biol. Chem., in press. 9. Gotoh, S., Nikolaev, N., Battaner, E., Birge, C. H. & Schlessinger, D. (1974) Biochem. Biophys. Res. Commun. 59,972-978. 10. Kramer, R. A., Rosenberg, M. & Steitz, J. A. (1974) J. Mol. Biol. 89,767-776. 11. Rosenberg, M., Kramer, R. A. & Steitz, J. A. (1974) J. Mol. Biol. 89,777-782. 12. Robertson, H. D. & Hunter, T. (1975) J. Biol. Chem. 250, 418-425. 13. Wellauer, P. K. & Dawid, I. B. (1973) Proc. Nat. Acad. Sci. USA 70,2827-2831. 14. Gralla, J. & Delisi, C. (1974) Nature 248,330-332. 15. Eron, L., Callahan, R. & Westphal, H. (1974) J. Biol. Chem. 249,6331-6338. 16. Eron, L. & Westphal, H. (1974) Proc. Nat. Acad. Sci. USA 71, 3385-3389. 17. Eagle, H. (1971) Science 174,500-503. 18. Anderson, C. W., Lewis, J. B., Atkins, J. F. & Gesteland, R. F. (1974) Proc. Nat. Acad. Sci. USA 71, 2756-2760. 19. Eron, L., Westphal, H. & Callahan, R. (1974) J. Virol. 14, 375-383. 20. Oberg, B., Saborio, J., Persson, T., Everitt, E. & Philipson, L. (1975) J. Virol. 15, 199-207. 21. Lewis, J. B., Anderson, C. W., Atkins, J. F. & Gesteland, R. F. (1974) Cold Spring Harbor Symp. Quant. Biol. 39,581-590. 22. Westphal, H., Eron, L., Ferdinand, F. J., Callahan, R. & Lai, S. P. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 575579. 23. Anderson, C. W., Baum, P. R. & Gesteland, R. (1973) J. Virol.
12,241-252. 24. Robertson, H. D. & Mathews, M. B. (1973) Proc. Nat. Acad. Sci. USA 70,225-229. 25. Berns, A. J. M., de Abreu, R. A., van Kraaikamp, M., Benedetti, E. L. & Bloemendal, H. (1971) FEBS Lett. 18, 159-163. 26. Portington, G. A., Kemp, D. J. & Rogers, G. E. (1973) Nature New Biol. 246,33-36. 27. Stevens, R. H. & Williamson, A. R. (1973) Proc. Nat. Acad. Sci. USA 70,1127-1131. 28. Swan, D., Aviv, H. & Leder, P. (1972) Proc. Nat. Acad. Sci. USA 69, 1967-1971. 29. Zelenka, P. & Piatigorsky, J. (1974) Proc. Nat. Acad. Sci. USA 71, 1896-1900. 30. Both, G. W., Banerjee, A. K. & Shatkin, A. J. (1975) Proc. Nat. Acad. Sci. USA 72, 1189-1193. 31. Altman, S. (1975) Cell 4, 21-29. 32. Bothwell, A. L. & Altman, S. (1975) J. Biol. Chem. 250, 1451-1463. 33. Winicow, I. & Perry, R. P. (1974) Biochemistry 13, 29082914. 34. Fuke, M. (1974) Proc. Nat. Acad. Sci. USA 71, 742-745. 35. Studier, F. W. (1973) J. Mol. Btol. 79,237-248. 36. Gaskill, P. & Kabat, D. (1971) Proc. Nat. Acad. Sci. USA 68, 72-75.
Cleavage of adenovirus messenger RNA and of 28S and 18S ribosomal RNA by RNase III (cell-free protein synthesis/gel electrophoresis of RNA fragments)
HEINER WESTPHAL AND ROBERT J. CROUCH Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014
Communicated by Bernhard Witkop, June 9,1975
Escherichia coli ribonuclease III cleaves adABSTRACT enovirus messenger RNA and mammalian 28S and ISS ribosomal RNA. Fragmentation is not random, but in each case a specific collection of products is generated. This points to the potential use of the enzyme as a tool for specific fragmentation of RNA. Cleavage by RNase III abolishes the capability of adenovirus messenger RNA to direct cell-free synthesis of virus polypeptides.
RNase III, an enzyme found in extracts of Escherichua coli, was originally thought to act exclusively on double-stranded RNA (1) generating oligonucleotides (2-4). Recently, however, considerable attention has been focused on the fact that precursors of both bacterial (5-8) and eukaryotic (9) rRNA, as well as the primary transcripts of early T7 DNA (6, 10, 11), are cleaved by the enzyme into fragments which are indistinguishable from those observed in vivo, implying a role for RNase III in the processing of RNA. At first sight, nearly complete degradation of doublestranded RNA and precise fragmentation of rRNA precursors or T7 transcripts are seemingly unrelated features of one enzyme. Yet, these two properties appear to copurify both genetically (5) and biochemically (4). It has been suggested that hairpin loops within the RNA molecule may be recognized by the enzyme (11, 12), possibly in conjunction with some signals conveyed by the primary structure next to the cleavage site (10, 8). Thus, base-paired regions of RNA may be the common denominators for recognition by RNase III. Helical structures have been most elegantly visualized in precursors of mammalian rRNA (13), and may be common as well to messenger RNA (14). A gradient of susceptibility of RNA sites to RNase III cleavage may be envisaged, with primary sequence and steric conformation determining the various degrees of exposure to enzymatic attack. While the signals for initial processing of large T7 transcripts and rRNA precursors are strong enough to elicit prompt response by the enzyme, more vigorous cleavage conditions may be needed for less exposed sites. Using three RNAs, adenovirus type 2 (Ad2) mRNA, 28S rRNA, and 18S rRNA, we will show here that unique patterns of fragments indeed result from cleavage by high doses of RNase III. MATERIALS AND METHODS RNase III was prepared as described (3). Upon isoelectric focusing of the material, one major band appears (R. J. Crouch, unpublished), which contains the RNase III activity. Ad2 mRNA. RNA was isolated from polysomes of KB cells, pulse-labeled with [3H]uridine at late stages of producAbbreviation: Ad2, adenovirus type 2. 3077
tive infection with Ad2. Poly(A)-containing RNA sequences were selected by oligo(dT)-cellulose chromatography (15). The preparation of [32P]mRNA followed the same protocol, except that cells were starved in phosphate-free medium (containing 2% dialyzed fetal calf serum) for a period of 2 hr preceding the pulse. A 2-hr incubation of 6 X 108 cells in 200 ml of medium, containing 7.5 mCi of carrier-free 32PO4, resulted in the preparation of 0.4 mg of mRNA with an initial specific radioactivity of 105 cpm/,ug. Less than 5% of the radioactivity of the Ad2 mRNA preparations was resistant to RNase A cleavage (for test conditions, see ref. 16). Cell-Free Protein Synthesis. Ad2 mRNA was translated in an S30 system of Krebs II ascites cells (15), with the following modifications. The assays contained up to 15 ng of exogenous mRNA per ll, 3.6 mM magnesium acetate, and 100 mM KC1. Translation products were analyzed by electrophoresis on 13% sodium dodecyl sulfate/polyacrylamide gels, and the radioactivity contained in bands of individual viral polypeptides was determined as reported (15). KB Cell rRNA. Growing cells were washed with, and resuspended in, phosphate-free medium containing 2% dialyzed fetal calf serum and 10 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), added to stabilize the pH (17). After 2 hr, 5 mCi of carrier-free 32pO4 was added to a culture containing 3 X 107 cells in 50 ml of medium, and 24 hr later cytoplasmic RNA (0.2 mg; 6 X 105 cpm/,ug) was prepared (15). Individual species of rRNA were gained by fractionation of cytoplasmic RNA on 1530% neutral sucrose gradients (16). Fractionated RNA was precipitated with ethanol, redissolved in H20 at 70 jig/ml, and stored for several weeks in liquid nitrogen, without noticeable degradation.
RESULTS Electrophoresis of RNA fragments cleaved from Ad2 mRNA and KB cell rRNA The observation of hairpin loops in ribosomal (13) and in Ad2 mRNA (J. Maizel, personal communication) and the suggestion that helical structures within a ribonucleic acid chain convey signals for RNase III attack (11, 12) prompted us to test the susceptibility of mammalian rRNA and of Ad2 mRNA to this enzyme. Based on the known preference of RNase III for doublestranded RNA (1), the enzyme dose was calibrated with the synthetic ribopolymer poly[r(A-U)] (3). An amount of RNase III sufficient to render >99% of the poly[r(A-U)] Cl3CCOOH-soluble did not change the capability of Ad2 mRNA to direct cell-free synthesis of Ad2 polypeptides. However, a further increase of the enzyme dose resulted in
3078
Biochemistry: Westphal and Crouch
Proc. Nat. Acad. Sci. USA 72 (1975) 1
2
3
4
5
6
8
7
4
28S
fs
AL.
18S
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pq
(c) 200
100
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.
6
0
.0
0.25 0.50 0.75 1.00 FRACTIONAL DISTANCE FROM BOTTOM
FIG. 1. Sucrose gradient analysis of degradation of Ad2 mRNA with RNase III and protection of degradation by duplex RNA. RNA (4.75 Mg) was incubated in 100 Ml containing 0.15 M KCl, 0.01 M MgCl2, 1 mM dithiothreitol, and 0.05 M Tris-HCl, pH 7.9, for 30 min at 37°. After an equal volume of a solution containing 10 M urea (Schwarz/Mann, ultrapure), 50 mM EDTA (neutralized with NaOH), and 2% sodium dodecyl sulfate was added, the reaction mixture was boiled for 1 min. Dilution with 133 MAl of H20 was followed by centrifugation at 4° for 220 min at 55,000 rpm in a 1530% (w/v) sucrose gradient, containing 0.1 M NaCl, 0.1 mM EDTA, 0.01 M Tris.HCl, pH 7.0, in a Spinco SW56 rotor. A parallel gradient contained KB cell [14C]rRNA as a size marker. Fractions were collected and radioactivity of aliquots was determined in scintillation fluid. (a) No enzyme, no duplex RNA; (b) 68 units of RNase III, 200 ,g of duplex RNA, isolated from replicative intermediates of bacteriophage MS2 RNA (a gift from Dr. Jerome Birnbaum, Merck Institute, Rahway, N.J.); and (c) 68 units of RNase III, no duplex RNA (see ref. 1 for definition of units).
the generation of Ad2 mRNA fragments that sedimented bea sucrose gradient (Fig. ic). A concern that it was not RNase III but a contaminating enzyme that was responsible for the observed decrease in sedimentation of the RNA was resolved by two types of experiments. First, addition of double-stranded RNA prevented the cleavage of Ad2 mRNA (Fig. lb). Second, under conditions of large excesses of Ad2 mRNA, complete protection of poly[r(A-U)] from degradation by RNase III was observed (data not shown). These results provided evidence that the sites cleaved in Ad2 mRNA and poly[r(A-U)] were attacked by the same enzyme, RNase III. Encouraged by the susceptibility of Ad2 mRNA to RNase III attack, we exposed mature mammalian ribosomal 28S and 18S RNA to comparable doses of enzyme and analyzed -the cleavage products by polyacrylamide gel electrophoresis. To allow for good resolution of all sizes of RNA between 28S and 4S, the gel was poured in two stages, with a gradient of acrylamide on the bottom and an agarose-acrylamide composite gel on top. The interphase between the two parts of the gel was clearly visible in the autoradiogram (Fig. 2). Ad2 mRNA incubated in the absence of enzyme displayed severtween 18S AND 4S rRNA markers in
.,
_
4S
FIG. 2. Polyacrylamide gel electrophoresis of RNase III-treated Ad2 mRNA and KB cell rRNA. Incubations of RNA and enzyme (see table below) were stopped by the addition of 1 volume (20 Mul) of 0.05 M EDTA, 10 M urea (Schwarz/Mann, ultrapure), 2% (v/v) 2-mercaptoethanol, 0.02% (w/v) bromophenol blue, 0.1 M NaPO4, pH 6.8,0.2% (w/v) sodium dodecyl sulfate, heated 1 min in boiling water, cooled to room temperature, and placed into preformed slots of a 1.5 mm (thickness) X 300 mm (length) X 160 mm (width) slab gel (35). The upper half of the gel contained 2.65% (w/v) acrylamide (Eastman) and 0.5% (w/v) agarose (Sigma), whereas the lower half of the gel consisted of a linear gradient of 3.5-20% acrylamide, stabilized by 0-10% (w/v) sucrose (Schwarz/ Mann, ultrapure). The concentration of N,N'-methylene-bisacrylamide (Eastman) was 0.08% (w/v) throughout the gel. Electrophoresis in 0.05 M NaPO4, pH 6.8, 0.1% sodium dodecyl sulfate, 2 mM EDTA (35) was at 15° for 17 hr at 350 V in a BioRad model 221 slab gel apparatus. The gel was briefly rinsed with water, dried under vacuum, and autoradiographed (35) for 2 days.
Gel slot 1 2 3 4 5 6 7 8
Substrate
Enzyme
RNA
ng
cpm
units
Ad2 mRNA Ad2 mRNA KB polysomal RNA KB 28S rRNA KB 28S rRNA
400 400 100 140 280 140 112 112
40,000 40,000 33,000 46,000 92,000 46,000 37,000 37,000
0 5.1 0 0 0.34 8.5
KB 28S rRNA KB 18SrRNA KB 18S rRNA
8.5 0
1-
Biochemistry: Westphal and Crouch M
b
a
c
d
Proc. Nat. Acad. Sci. USA 72 (1975)
3079
e
8
pVI
HEXON
~75 z
0
55c-
------
-------
---------
Co ,0-
Adma.
LL25
AW S.
-
AeW,
0
10
20 30 40 ENZYME CONCENTRATION (U/ml)
50
FIG. 4. Loss of translation capacity of individual Ad2 mRNAs as a function of RNase III cleavage. The radioactivity contained in the polypeptide II and pVII bands of columns a-e, Fig. 3, was determined (15), and expressed as percent fraction of the control (100% refers to 3020 cpm of polypeptide II and 3855 cpm of polypeptide pVII, column a, Fig. 3).
pVIIVil-
AMM 4I
i
11*
Alw_
... o*
.pm ~~~~~~~-WI
... .
L _~~~~~~O
VI-w,_
FIG. 3. Cell-free translation of RNase Ill-treated Ad2 mRNA. RNA (260 ng) was incubated in 10-gl assays with (a) 0, (b) 0.08, (c) 0.19, (d) 0.28, or (e) 0.53 unit of enzyme. No RNA or enzyme was added to a control assay (f). At the end of the incubation period, 5-,ul aliquots of each assay were added to 55 ,l of translation mixture. Translation products obtained in this reaction (% of the reaction mixture) were analyzed by electrophoresis through a 13% sodium dodecyl sulfate/polyacrylamide gel (15) (thickness 1.5 mm, length 300 mm, width 160 mm) at room temperature for 15 hr at 200 V. The figure represents an autoradiogram of the dried gel, depicting 35S-labeled polypeptide bands. Roman numerals refer to individual polypeptide components (23) of an adenovirion preparation (15), added as a size marker (M).
al bands which, although visible in the original autoradiopoorly resolved in the photograph (Fig. 2, column 1). Nevertheless, the shift of RNA radioactivity toward greater electrophoretic mobility after enzyme treatment (Fig. 2, column 2) was obvious. Much more distinct were the patterns of RNA fragments resulting from the treatment of purified 28S rRNA (Fig. 2, column 4) with RNase III. Whereas, a low dose of enzyme produced fragments that migrated in several bands, some between 28S and 18S rRNA markers, one near the bottom of the gel (Fig. 2, column 5), a high amount of enzyme resulted in further fragmentation, as evidenced by a number of bands displayed in the lower half of Fig. 2, column 6. No radioactivity remained at the origin, indicating that all 28S rRNA had been cleaved. On the other hand, 18S rRNA apgram, were
peared more resistant to RNase III attack, with only a portion of the RNA cleaved into fragments which formed a pattern distinct from that of 28S rRNA cleavage products. Translation of RNase Ill-treated Ad2 mRNA A number of virion polypeptides are synthesized in cell-free systems directed by late Ad2 mRNA (15, 18, 19, 16, 20-22). In an attempt to correlate the observed fragmentation of the RNA by RNase III with its biological activity, we translated enzyme-treated Ad2 mRNA in an S30 extract of Krebs II ascites cells. This cell-free system had a considerable background of endogenous protein synthesis in the absence of added mRNA, as demonstrated in column f of Fig. 3, displaying the electrophoretic pattern of polypeptides synthesized in the control assay. Polypeptide migration was toward the bottom of the gel, separating polypeptides in the size range of >105 to about 104 daltons. Addition of 30 ng of Ad2 mRNA to the cell-free system resulted in the synthesis of a number of virion protein components, some of which could easily be discerned (column a, Fig. 3) using adenovirion polypeptides (column M) as a reference. Treatment of Ad2 mRNA with increasing doses of RNase III prior to cellfree translation led to the gradual disappearance of the polypeptides specified by Ad2 mRNA (columns b through e). Yet, the sensitivity of in vitro expression to enzyme attack varied considerably among individual virus-specific polypeptides. For instance, the amount of RNase III required for a 50% reduction in the synthesis of polypeptide pVII, the precursor (23) of core protein VII, was more than twice that needed to reduce hexon (= polypeptide II) synthesis to the same level (Fig. 4). Ad2 mRNAs coding for individual components of the virus particle have been partially separated by sucrose gradient centrifugation (18, 22, 20). The size of some of these mRNAs, estimated from their sedimentation velocities, exceeds that required to specify the respective polypeptide. For instance, pVII, a small polypeptide of 20,000 molecular weight (23), is expressed by an RNA of perhaps as much as five times (18) the pVII code size. This has led to the suggestion that the RNA may be polycistronic, i.e., coding for
3080
Biochemistry: Westphal and Crouch
Proc. Nat. Acad. Sci. USA 72 (1975)
more than one polypeptide (18). If so, treatment of Ad2 mRNA with RNase III might generate fragments that retain the coding capacity for an entire virus polypeptide, provided the enzyme does not destroy signals required for proper translation. Accordingly, we treated Ad2 mRNA with increasing amounts of RNase III (corresponding to assays a through e, Fig. 3), fractionated the cleavage products by sucrose gradient centrifugation, and tested the RNA contained in individual gradient fractions in translation assays. The sedimentation profiles of the Ad2 mRNA (Fig. 5) showed a gradual shift of radioactivity from a position between 28S and 18S rRNA (assay a = Ad2 mRNA incubated in the absence of RNase III) to a position close to 4S (assay e = highest enzyme concentration). RNA contained in individual fractions of the sucrose gradient gave rise to the cell-free translation products displayed in the electropherograms at the left of Fig. 5. The virus-specific protein that could be most readily discerned from the background of endogenous material is polypeptide II or hexon, visible as the top band in the columns corresponding to fractions 7-12 of panels a-d. Most of the mRNA giving rise to polypeptide II appeared to be contained in fractions 9-10, in quantities inversely proportional to the amounts of enzyme used. Like polypeptide II, none of the smaller virus-specific polypeptides, such as polypeptides III, IV, or pVII, varied in position in the sucrose gradient, no matter which concentration of RNase III had been used (note that pVII is present, albeit poorly resolved, in panels a and c); moreover, no additional polypeptide bands were detectable among the translation products of RNase III-treated Ad2 mRNA. Thus, we were unable to detect fragments of Ad2 mRNA that retained their ability to specify cell-free translation products.
0 -D
3. 5- 7- 9- 11-13-15-17-
4 6 8 10 12 14 16 18
DISCUSSION Two distinct types of eukaryotic RNA, adenovirus messenger RNA and KB cell ribosomal RNA, were found susceptible to attack by RNase III. With each RNA, unique patterns of fragments were observed. Certain RNA sites were more sensitive to cleavage than others, as evidenced by the appearance of large fragments of 28S rRNA which were further cleaved by a higher dose of enzyme. These findings are compatible with the postulate that RNase III, an enzyme with known specificity for double-stranded RNA, attacks helical structures in each of the RNAs tested, and that a gradient exists of susceptibility of individual RNA sites to enzymatic cleavage. If so, RNase III, as well as endonucleases with comparable properties may assume the role of tools for RNA analysis in a manner analogous to that of restriction enzymes in the analysis of DNA. For instance, the enzyme might become important for the analysis of regions of nonhomology distinguishing closely related RNAs, for RNA sequence determinations, for examining new pathways of RNA processing, and for isolating RNA segments of special interest, such as the variable region within an immunoglobulin RNA chain. RNase III degrades double-stranded RNA into fragments 15-20 nucleotides in length (2-4). Possibly, helical structures within a single RNA chain are degraded in a similar fashion. For instance, an RNA chain containing a single helical region susceptible to enzyme attack would thus be cleaved into two polynucleotide fragments corresponding to the RNA segments on either side of the helix. An oligonucleotide, corresponding to the helical region, would also be produced.
5
10
15
FRACTION NUMBER FIG. 5. Sucrose gradient centrifugation and cell-free translation of Ad2 [3H]mRNA cleaved by RNase III. Aliquots (75 gl) of RNase III assays a-e (Fig. 3) were sedimented through sucrose gradients (see Fig. 1), and the radioactivity of 0.25 volume of each gradient fraction was determined in scintillation fluid. The RNA sedimentation profiles appear at the right side-of the figure. The remainders of the sucrose gradient fractions were combined, as indicated at the left of the abscissa, adjusted to 0.15 M KCl, and precipitated by ethanol, together with 4 ,ug of carrier rabbit tRNA (General Biochemicals). The samples were centrifuged overnight at -20° in a swing-out rotor at 1800 X gmax, washed with 70% (v/v) ethanol in water, recentrifuged for 30 min, dried, and taken up in 60 ,ul of translation mixture. The 35S-labeled products of the cellfree translations were electrophoresed on standard 13% sodium dodecyl sulfate/polyacrylamide gels (15). The autoradiograms of these gels, shown on the left side of the figure, include adenovirion marker proteins (M), aliquots of the translation assays of Fig. 3 (A), and translation assays lacking exogenous mRNA (B). Roman numerals at the left ordinate refer to individual adenovirus poly-
peptides (see Fig. 3).
We have made no attempts to account for fragments of oligonucleotide size and, therefore, have not data to support the mechanism of cleavage envisioned here. In any event, RNase III treatment of Ad2 mRNA did not generate significant amounts of radioactivity remaining on top of the sucrose gradients (Fig. 5). This indicates that the fraction of RNA, if any, that is degraded to oligonucleotide size must be small, or, conversely, that most or all of the RNA is represented in fragments of polynucleotide size. A strong link between the structural integrity of messenger RNA and its biological activity is indicated by the find-
Biochemistry: Westphal and Crouch ing that exposure of Ad2 mRNA to RNase III destroyed its capability of directing cell-free synthesis of virus-specific polypeptides, a result which could explain the inhibitory effect of high concentrations of RNase III on the cell-free translation of encephalomyocarditis RNA (24). Evidence has accumulated which suggests that eukaryotic mRNA contains more information than is necessary to encode for the cognate protein (25-29, 36). The function(s) of such extra RNA is, as yet, unknown. It is conceivable that the rapid inactivation of Ad2 mRNA to act as a message in a cell-free system after treatment with RNase III reflects a preference of the enzyme for sites present in the extra RNA regions and not an attack in the coding sequence. Certainly if RNase III action separates the coding sequence from any signal required for translation, the RNA would be inactivated without the necessity of a cleavage in the region of the encoded message. Recently, Both et al. (30) have proposed that the 5'-terminal 7-methyl guanosine is necessary for efficient translation of vesicular stomatitis virus and reovirus mRNA. If the Ad2 mRNA has a similar requirement, simply removing a few nucleotides from the 5' terminus would render the mRNA inactive for in vitro protein synthesis. The greater the distance the coding sequence is from the 5'-terminal methylated nucleotide, the greater is the chance for separation of the translated sequence and the 5' terminus. Even though the mRNA for pVII sediments as if the RNA is five times (18) the pVII code size, we find the mRNA, after treatment with RNase III, in a translatable form only in the region of the sucrose gradient in which untreated pVII mRNA is found. Such an observation is consistent with either a direct inactivation of the pVII mRNA by nucleolytic action in the coding sequence or by a severing of the translatable sequence from a structure or sequence necessary for proper translation. In light of this consideration, the preservation of translatable Ad2 mRNA sequences after RNase III attack would seem indeed unlikely. The in vitro synthesis of the hexon polypeptide is more affected by RNase III action than that of pVII, the precursor to core polypeptide VII. Hexon mRNA sediments ahead of pVII mRNA in sucrose gradients. This suggests that, as the larger target, it is more vulnerable to RNase III attack. Alternatively, or in addition, the Ad2 mRNA preparation may contain more copies of pVII mRNA than of hexon mRNA, or hexon mRNA may harbor sites more sensitive to RNase III than those of pVII mRNA. The list of RNases of both prokaryotic (31) and eukaryotic (32, 33) origin with limited and specified endonucleolytic action is growing rapidly. Even RNase T1, when tested under restricted conditions (34), may be included in this list. This area of research may be expected to bring deeper insight into the various pathways of RNA processing and degradation and their regulation in vivo while generating more refined methods for RNA analysis in vitro. We thank Dr. J. J. Dunn for helpful discussions, Dr. F. -J. Ferdinand for Ad2 [ssP]mRNA, Mrs. S.-P. Lai for superb technical assistance, Drs. H. D. Robertson, J. J. Dunn, D. Ginsburg, and J. A. Steitz for making their results available to us prior to publication, and Dr. Jerome Birnbaum for his generous gift of duplex RNA.
Proc. Nat. Acad. Sci. USA 72 (1975)
3081
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