Rapidly labeled RNA associated with ribosomes of tobacco leaves infected with tobacco mosaic virus

VIROLOGY

39, 893-900

Rapidly

(1969)

Labeled

RNA

Associated

Leaves

Infected

with

with Tobacco

Ribosomes Mosaic

of Tobacco Virus’

I’. BABOS Department

of Botany,

Washington Accepted

University, July

St.

Louis,

Missouri

63130

30, 1969

Sucrose density gradient analysis of the RNA extracted from ribosomes of actinomycin D-treated and labeled tobacco leaf tissue infected with TMV showed that the radioactivity was incorporated approximately equally in 18 S RNA and in a small amount of low-molecular weight RNA of high specific activity. When ribosomes were dissociated with EDTA, the radioactivity was similarly approximately equally apportioned between low-molecular weight, highly labeled RNA and nucleoprotein particles sedimenting as the 40 S ribosomal subunit. INTRODUCTION

In a previous paper (Babos and Shearer, 1969) it was reported that infected leaves exhibited an AMDZ-resistant radioactivity in excess of that incorporated in TMV-RNA, most of which sedimented in sucrose density gradients at the position occupied by the light, 18 S, rRNA. This paper describes the results of attempts to distinguish between normal 18 S rRXA and the virus-induced, rapidly labeled RNA having the same sedimentation rate. For that purpose, ribosomes were isolated and their RNA was analyzed as such or as nucleoprotein particles in linear and part-linear sucrose density gradients. MATERIALS

AND

METHODS

The materials used and the methods employed were as described previously (Babos and Shearer, 1969). To extract ribosomes, 1 g of tobacco leaf tissue was ground at 4” in 3 ml of buffer consisting of 0.1 M Tris, containing 0.5 M sucrose, 0.01 ild KCl, 0.01 k? 1 This investigation was supported by research grant GB-3113 and GB-6599 (renewal) from the National Science Foundation. 2 Abbreviations: TMV, tobacco mosaic virus; RNA, ribonucleic acid; rRNA, ribosomal RNA; EDTA, ethylenediaminetetraacetic acid; TCA, trichloroacetic acid; Tris, tris(hydroxymethyl)aminomethane; AMD, actinomycin D; TK buffer, Q.025 M Tris, 0.01 M KCl, pH 7.6.

magnesium acetate, and 0.01 1M mercaptoethanol, and adjusted with HCl to pH 7.6 at room temperature. Acid-washed sand at 0.3 g per gram of tissue wm also added to facilitate disruption of the tissue. Grinding was done in a mortar with a pestle for 3 min for the first gram of tissue and 2 min for each additional gram. The homogenate was centrifuged at 40,000 g for 10 min to pellet particulate matter except ribosomes. The supernatant was usually clear yellow, but occasionally a faint green tinge indicated the rupture of some chloroplasts. The green matter of the pellet was firm so the supernatant was decanted without upsetting it and passed through one layer of cheesecloth to retain any material that was floating on the supernatant. The supernatant was then centrifuged at 102,000 g for 90 min to pellet the ribosomes and the virus in the case of infected tissue. The pellet was gently suspended in TK buffer containing 0.01 M magnesium acetate and was clarified by centrifuging at 10,000 g for 2 min. Part-linear sucrosedensity gradients were made as described in the legend of Fig. 4. RESULTS

Fractionation of Ribosomes and rRNA in Linear Density Gradients To investigate the incorporation of label in ribosomesof infected and healthy tobacco 893

BABOS

Healthy

Infected

AMD

too 75 5 4 50

25

0

10

20

10

20 FRACTION

10

20

10

20

30

NO

FIG. 1. The effect of actinomycin D (AMD) on the incorporation of uracilJ% in ribosomes of TMVinfected and healthy tobacco leaf tissue. One gram of tissue was exposed to AMD at 50~g/ml for 2 hours, before labeling with 1.25 &i/ml uracil-W for 2 hours. Ribosomes and virus were extracted simultaneously by centrifuging the clarified sap (40,000 g for 10 min) at 102,000 g for 90 min. The pellet was dissolved in 1 ml of TK buffer containing 0.01 A4 magnesium acetate and analyzed in a linear sucrose density gradient in TK buffer with 0.01 M magnesium acetate. The peak of optical density at fraction 8 is TMV, at fraction 18 is the ribosome (Babos, 1966). O---O, Optical density at 2GO mp; 0 - - - -0, radioactivity.

leaves in the presence of AMD, tissue was exposed to ARID at 50 fig/ml for 2 hours before labeling with uracilJ4C for l-2 hours, and the ribosomes were extracted and analyzed in sugar gradients. Figure 1 shows that A:\ID inhibited the incorporation of label into ribosomes of healthy leaves, and much less so into ribosomes of infected leaves. Relative to the amount of radioactivity incorporated into virus particles, there was a significant amount of radioactivity in the ribosomal peak of the AMD-treated tissue. Similar results were obtained when the high speed pelleting of ribosomes before sugar gradient analysis was omitted (Fig. 2). In this case, the sap was clarified by centrifuging at 40,000 g for 10 min and layered on the sugar gradient. On centrifugation the ribosomal peak sedimented ahead of the optical density front caused by the diffusion of colored matter in the clarified sap. This method is not only more rapid, but avoids further manipulation of ribosomes. When the ribosomal pellet, or the pooled fractions corresponding to the ribosomal

peak as in Fig. 2, was extracted with phenol and the resulting RNA was analyzed in sugar gradients, it was found that only a fraction of the radioactivity corresponded to the two high molecular weight rRNA components (Fig. 3). Very little radioactivity was present in the RNA of ribosomes from AMD-treated healthy leaves, as expected from the ribosomal labeling, whereas the RNA from ribosomes of AMD-treated infected leaves contained considerable radioactivity in all parts of the gradients which were either of equal or lower sedimentation rate than the 18 S rRNA. On the other hand, the RNA extracted from the ribosomes of infected control leaves contained much more radioectivity in all positions of the gradient including the rRNA components. The RNA extracted from the ribosomes of healthy cantrol leaves was much like that of the infected control leaves. The RNA component of the highest specific radioactivity sedimented near the top of the gradient, in both AMDtreated and control infected leaves. When the amount of RNA and the radioactivity

RIBOSOMES

IN TMV-INFECTED

LEAVES

895

1

Infected-

Infected

Healthy-

AMD

AMD

,

i

2.5

L

,

,

0

10

20

0

10

20

0

10

20

FRACTION NO. 2. The effect of actiuomyein D (AMD) on the incorporation of uracil-14C in ribosomes of TMVinfected and healthy tobacco leaf tissue. One gram of tissue was exposed to AMD at 50 pg/ml for 2 hours before labeling with 1.25 pCi/ml uracil-W for 2 hours. The tissue was ground in Tris buffer as described in text and the sap was clarified by centrifuging at 40,000 9 for 10 min. Four milliliters of the clarified sap were layered on 27 ml of a G-30% linear sucrose density gradient in TK buffer with 0.01 M magnesium acetate and centrifuged at 25,000 rpm for 5 hours. The optical density peak at fraction 3 is TMV; and at fraction 17, the ribosome. a---@, Optical deusity at 260 mr; 0- - - -0, radioactivity. FIG.

recovered from the ribosome peak, such as shown in Fig. 2, mere determined, it was found that 84% of the RNA was recovered, but only 45 % of the radioactivity, indicating that, some of the highly labeled low molecular

weight RNA was probably lost during the phenol extraction and purification of RNA. The existence of several RNA components in the optical density profiles of RNA extracted from ribosomes of infected leaves, and their similarity with the RXA extracted from healthy leaves, makes impossible the assignment of any component as virus specific. However, on suppressionof the cellular RNA synthesis by AMD, the radioactivities of the 25 S and 18 S RNA were disproportionally affected. The ratio of radioactivity of the 18 S to 25 S RNA became several times higher in ARID-treated infected leaves than

in the untreated

ones.

Fractionation

of Ribosomes in Part-Linear

Gradients Analysis of the total RNA extracted from ribosomes of infected leaves revealed differences in the incorporation of label by some RNA components, but no qualitative difference was observed. A method was sought that would preserve the structural integrity of the ribosome, or the ribosomal subunits, and yet separate any ribosome-bound radioactivity as RNA or nucleoprotein. To achieve this, use was made of the property of EDTA of dissociating RNA bound to ribosomes(Marbaix et al., 1966; Hadjiolov, 1966; Parish et al., 1966; Perry and ECelley, 1968; Henshaw, 1968) and of part-linear sucrose gradients to separate the dissociated components. Details of the method and its application to separation of rapidly labeled

UAROS

Infected-AMD

Healthy-AMD t

0

10

20

30 0

10 FRACTION

FIG. 3. The effect, of actinomycin D (AMD) from ribosomes of healthy and TM\‘-infected pg/ml for 2 hours, and labeled with 1.25 &,X/ml Fig. 2. Fractions corresponding to the ribosome was analyzed in a 5-20,, O’ linear sucrose density EDTA, pH 9.5. The optical density peaks at respectively. @---a, Optical density at 260

20

: 30 0

10

20

30

NO.

on the incorporation of uracil-14C in RNA extracted tobacco leaf tissue. Tissue was exposed to AMD at 50 uracil-“C for 2 hours. Ribosomes were isolated as in peak were pooled, and the RNA, extracted with phenol, gradient made in 0.1 M glycine, 0.1 M KCl, and 0.01 M fractions 10 and 17 correspond to 25 S and 18 S rRNA, rnp; O- - - -0, radioactivity.

RNA from ribosomes of tobacco leaf tissue will be presented elsewhere. Briefly the method allows the “washing” of ribosomes while sedimenting through a critical Mg2+ concentration, the disruption of Mg2+-mediated bonds to release matter so bound to the ribosomes, and the separation of ribosomal subunits, all in one operation in a sucrose gradient that is processed as usual. The ribosomal and ribosome-associated components remain essentially in their native state, and this avoids degradation and loss of RNA, especially of low molecular weight, during the lengthy procedure of extracting RNA with phenol. Figure 4 compares the optical density and radioactivity profiles of ribosomes extracted from healthy and infected tobacco leaves and centrifuged in linear and part-linear sucrose density gradients. In the linear gradients the radioactivity profiles correspond to the optical density profiles. The gradual increase in optical density toward the peak from the denser part of the medium and the correspondingly higher radioactivity represent small aggregates of ribosomes, possibly broken polyribosomes. The ribosomes from infected leaves had rather higher spe-

cific activity in the experiment quoted, but this was not always the case. The part-linear gradients show that when the ribosomes reached the portion of the gradient containing EDTA they released a considerable amount of their radioactivity which remained near the beginning of the EDTA solution. The sudden increase in sucrose concentration helped to confine the released radioactivity within a fairly sharp band. The radioactivity of this band in tobacco leaves was shown to be highly labeled RNA and some transfer RNA (Babos, manuscript in preparation) and not unspecific attachment of label to ribosomes. The reasons given are: TCA-insolubility, degradation by ribonuclease, disappearance on prolonged “chase” in cold medium, increase with labeling time, and disappearance after AMD treatment. The ribosome itself was split in components sedimenting with two sedimentation rates. These correspond to the two ribosomal subunits of 60 S and 40 S sedimentation coefficients, although they sedimented fractionally slower than the corresponding subunits released from the ribosome in the absence of Mg2+. It was shown for bacterial (Gesteland,

IlIBOSOMES

TMV-INFECTED

Infected

Infected

0

IN

IO

20

10

20

Healthy

Healthy

a

b

10

30 FRACTION

597

LEAVES

20

10

20

30

NO.

FIG. 4. Sedimentation of ribosomes extracted from TMV-infected or healthy tobacco leaf tissue in linear (a) and part-linear (b) sucrose density gradients. Ribosomes were extracted from 4 g of tissue labeled wit,h 1.8 rCi/ml uracil-W for 2 hours. The ribosomal pellet was suspended in 2.5 ml TK buffer containing 0.01 M magnesium acetate and clarified by centrifuging at 10,000 9 for 2 min. One milliliter was layered on 29 ml of a 5-20% linear sucrose density gradient in TK buffer containing 0.01 M magnesium acetate and centrifuged at 25,000 rpm for 3 hours. Also 1 ml was layered on a part-linear sucrose density gradient made with 24 ml of IO-207& sucrose in TK buffer containing 0.01 M EDTA, 1 ml of 57, sucrose in TK buffer, and 3 ml of 2.5% sucrose in TK buffer containing 0.001 M magnesium acetate, successively. The gradient was centrifuged at 25,000 rpm for 8 hours. The ratio of distances from fraction 25 of the two major optical density peaks is approximately 3:2, as expected of the 60 S and 40 S ribosomal subunits. a--@, Optical density at 260 mr; 0- - - -0, radioactivity.

1966) and for mammalian ribosomes (Tashiro and Siekevitz, 1965; Defilippes, 1967) that this is due to partial “unfolding” of the subunit structure with an increase of the hydrodynamic volume. The specific activity of the small ribosomal subunit was higher than that of the large one, as could be expected from the analysis of the ribosomal RNA (Fig. 3). No qualitative difference was observed between the gradients of infected and healthv leaves. However, when infected and healthy tissues were treated with AMD at 50 pg,/ml for 2 hours before labeling and the extracted ribosomes were analyzed on part-linear gradients, quite different radioactivity patterns were observed with respect to the total radioactivity incorporated at all positions of the gradient, and the relative labeling of individual components (Fig. 5). The relative labeling of the ribosomal sub-

units of AMD-treated healthy tissue was essentially similar to that of the untreated control (Fig. 4). In the case of infected AMD-treated tissue the ratio of radioactivity of the 40 S to that of the 60 S subunit was several times higher than in the infected untreated control. This ratio describes this situation well, as it is independent of the actual labeling of the two components. The radioactivity of the RNA which dissociated from the ribosomes (Fig. 5, fractions 20 to 25) was more than three times higher in the AMD-treated infected tissue than in the healthy control. On prolonged centrifugation this radioactivity peak split into two peaks (Fig. 6). In Fig. 5 a representative experiment is quoted. However, the quantitative relationships of the different peaks of radioactivity varied in different experiments although all

BAKE

898

Infected

d d

Iiealthy

250

.8

200

.6

150 z

.4

100

.2

50

u”

0

10

20 ’ 30 FRACTION

0 NO.

10

20

’

30

FIG. 5. Distribution of optical density at 260 rnF (+--a), and radioactivity (O- - - -0) profiles of part-linear sucrose density gradients of ribosomes extracted from 1 g of TMV-infected or healthy tobacco leaf tissue exposed to AMD at 50 pg/ml for 2 hours and labeled with 20 &i/ml 32P for 2 hours. Similar profiles were obtained when the tissue was labeled with uraoil-14C. The gradients were centrifuged for 8 hours; other experimental details were as in Fig. 4.

.6 .5 d d

.4 .3 .2 .l

0

10

20

30

10 FRACTION

20

30

10

20

30

NO.

FIG. 6. Distribution of optical density at 260 rnp (O--O), and radioactivity (0- - - -0) profiles of part-linear sucrose density gradients of ribosomes extracted and processed separately from 1 g each of TMV-infected tobacco tissue, treated with actinomycin D at 50 rg/ml for 2 hours and labeled with 5 &X/ml 32P for 2 hours. The gradients were centrifuged for 10 hours; other experimental details were aa in Fig. 4.

RIBOSOMES

IN

TMV-INFECTED

experiments were done between 2 and 3 days after inoculation. To determine how much of the variation was due to experimental error, three different samples of the same infected tissue were separately processed. Figure 6 shows that there is good agreement among them, so the variation should normally be caused by the different physiological states of the leaves at the time of each experiment. DISCUSSION

The ribosomal characteristics of the young tobacco leaf are similar to those of the “active” ribosome of bacterial cells (Tissieres, et al., 1960) and to the monosomeof eucaryotic cells (Staehelin et al., 1964; Loening, 1962), which is a single ribosome with a short length of “messenger” RNA, 30-50 nucleotides long (Takanami et al., 1965), even in ribonuclease-treated ribosomes, together with nascent protein and transfer RNA. The lack of coincidence between the optical density and radioactivity profiles of sucrose density gradients of RNA extracted from ribosomesshowsthat a large proportion of label is incorporated into RNA which is distinct from ribosomal RNA. In this respect, there was no difference between healthy and TJIV-infected tissue 2-3 days after inoculation, for labeling periods of up to 2 hours. This suggeststhat either there is no alteration of the ribosomal status quo in the infected tissue, or the participation of viral RNA, if any, in the ribosome complex is done at the expense of normal RNA. The excessAl/ID-resistant radioactivity observed in the ribosomes of infected tissue suggests that the latter explanation may be more plausible. Treatment of tobacco leaf ribosomes with EDTA further substantiated that the highly labeled RNA is extraneous to the ribosome. The large AYID-resistant radioactivity of the 18 S RNA of infected tissue, when total leaf RNA is extracted with phenol (Babos and Shearer, 1969) could be the result of complexing of the rapidly labeled RNA with 18 S ribosomal RNA (Staehelin et al., 1964; Hayes et al., 1966). However, in order for this RNA to remain attached to the small ribosomal subunit in the presenceof EDTA, it must be covalently

LEAVES

s99

linked to it, or be an integral part of the structure of the subunit (Hadjiolov, 1967). Perhaps it is a special nucleoprotein particle with similar sedimentation behavior as the normal ribosomal subunit. It is tempting to suggest that the characteristics of labeling of cytoplasmic ribosomes of AMID-treated infected leaves are due to their involvement in the synthesis of viral protein. Because we know little of the effect of AND on the metabolism of healthy or infected tobacco cells, such a conclusion would be premature. Experiments are now in progress to determine the relationship, if any, of the low-molecular weight, rapidly labeled RNA, and the 18 S RNA with TMV-RNA or its complementary strand. REFERENCES BABES, P. (1966). The ribonucleic acid content of tobacco leaves infected with tobacco mosaic virus. Viroiogy 28, 282-289. BABOS, P., and SHEARER, G. B. (1969). RNA synthesis in tobacco leaves infected with tobacco mosaic virus. Virology 39, 286-295. DEFILIPPES, F. M. (1967). Sucrose gradient sedimentation of dissociated HeLa cell ribosomes. Biochint. Biophys. Acta 145, 337-352. GESTELAND, It. F. (1966). Unfolding of Escherichia coli ribosomes by removal of magnesium. J. Mol. Biol. 18, 356-371. HADJIOLOV, A. A. (1966). Studies on the turnover and messenger activity of rat-liver ribonucleic acids. Biochim. Biophys. Acta 119, 547-556. HADJIOLW, A. A. (1967). Ribonucleic acids and information transfer in animal cells. Progr. Nucleic Acid Res. 7, 195-242. HAYES, D. H., HAYES, F., and GUERIN, M. F. (1966). Association of rapidly labelled bacterial RNA with ribosomal RNA in solutions of high ionic strength. J. Mol. Riot. 18,499-515. HENSHAW, E. C. (1968). Messenger RNA in rat liver polyribosomes: Evidence that it exists as ribonucleoprotein particles. J. Mol. Biol. 36, 401411. LOENING, U. E. (1962). Messenger ribonucleic acid in pea seedlings. Nature 195, 467-469. MARLIAIX, G., BURNY, A., HUEZ, G., and CHANTRENNE, H. (1966). Base composition of messenger RNA from rabbit reticulocytes. Biochim. Biophys. Acta 114, 404406. PARISH, J. H., KIRBY, K. S., and KLUCIS, E. S. (1966). Rapidly labelled, low molecular weight RNA from rat liver cytoplasmic ribonucleoprotein particles. J. Mol. Biol. 22, 393-395.

900

BrlBOS

P. R., nrd KELLEY, I). E. (1968). MCSSCIIger RNA protein complexes s11d newly synthesized ribosomal subunits: Analysis of free pnrticles and components of polyribosomes. J. Mol. Bioi. 35,37-59. STAEHELIN, T., WETTSTEIN, F. O., C)um, II., am1 NOLL, H. (1964). Determination of the coding ratio based on molecular weight of messenger ribonucleic acid associated with ergosomes of different aggregate size. Nature 201, 264-270. PERRY,

TAICASAJII, >I., YAK, Y., and JUKES, T. II. (1965). Studies on the site of ribosomal binding of f2 bacteriophage RNA. J. Mol. Biol. 12,761-773. T.KSIII~O, Y., nrd SIEKEVITZ, P. (1965). Ultracentrifugal studies on the dissociation of hepatic ribosomes. J. Mol. Biot. 11, 149-165. TISSIERES, 9., SCHLESSINGER, II., and GROS, I?. (1960). Amino acid incorporation into proteins by Escherichia coli ribosomes. Proc. Natl. Acad. Sci. TT.S. 48, 1450-1463.