Entrance of newly formed messenger RNA and ribosomes into HeLa cell cytoplasm

J. Mol. Biol. (1965) 11, 187-201

Entrance of Newly Formed Messenger RNA and Ribosomes into HeLa Cell Cytoplasm M. GIRARD,t H.

LATHAM ,

S. PENMAN

AND

J . E.

DARNELL

Department of Biology, .Massachusetts Institute of Technology Cambridge 39, Mass., U.S.A . (Received 25 October 1963, and in revised form 13 August 1964) Newly formed ribosomal RNA is sho wn t o appear in the H eL a cell cy toplas m first in precursor particles, then in polyr ib osome s and finally in free 74 s r ibosomes. The implication of these find ings is dis cussed.

1. Introduction Previous work on the RNA of HeLa cells, a strain of human cells capable of ind efinite rapid growth under defined conditions, has shown the following. (1) The majority of 32p or He which is rapidly incorporated into RNA is found in the cell nucleus in high molecular weight precursors of ribosomal RNA (Scherrer & Darnell, J962; Scherrer, Latham & Darnell, 1963). If actinomycin D is used to inhibit RNA synthesis after the cells have been previou sly lab eled for 30 minutes, the high molecular weight nuclear material is lar gely conserved (at least 60% ) and conver ted to the proper size for ribo somal RNA. Although the maj ority of this radi oacti ve RNA remains in the nucleus, there is a transfer of some to the cytoplasm in th e form of completed ribosomes (Girard, Penman & Darnell, 1964). That which remains in the nucleus, although possessing the proper sedimentation characteristics (28 sand 16 s) for ribosomes, is not in completed 74 s particles. (2) In addition to the rapidly lab eled nu clear RNA which becomes stabiliz ed as 28 sand 16 s R-RNAt after actinomycin treatment, there is ano th er fraction (about one-third of a 30-minute uridine label) which is d egraded within 30 minute s t o acidsoluble material (Scherrer , Latham & Darnell, 1963). Thi s fra ction may be identical to the unstable nu clear RNA first described by H arris and his collab orators (Harris, 1963; Harris, Fisher , Rodgers, Spencer & Watts, 1963). The nature of this unstable nuclear RNA is unknown. (3) A very small proportion of rapidly labeled RNA can be identifi ed as M-RN A in the cell cytoplasm (Penman , Scherre r, Beck er & Darnell, 1963). Thi s l\I-RN A is distinguishable from R·RNA on the ba sis of sediment at ion properties and a " D NAlike" base ratio and was recognized by virtue of its associat ion with groups of ribosomes (polyribosomes) which are the active site s of protein synthesis in the cell cytoplasm (Oierer, 1963; Warner, Knopf & Rich, 1963; Wettstein, Staehelin & Noll, 1963).

t Present add ress: Albert E inst ein College of Medicin e, Yesh iva Univer sit y , N ew Y ork 61, New York , U.S.A . t Abbreviations used : S-RNA, soluble ribonucleic a cid; M·RNA, m essenger ri bonucleic a cid : R-RNA, ribosomal ribonucleic ac id ; DOC, sodium deoxycholate. 187

188

M. GIRARD, H. LATHAM, S. PENMAN AND J. E. DARNELL

This series of experiments has been continued by following the kinetics of appearance of ribosomal and messenger RNA in various structures present in cytoplasmic extracts. It has been found that two structures smaller than 74 s ribosomes ("native" particles) first become labeled with R-RNA. These precursors to finished ribosomes are compared to the dissociation products of ribosomes produced either by low Mg 2 + ion concentrations (low Mg 2 + particles) or by treatment of ribosomes with EDTA particles. In addition to the studies on the sub-ribosomal particles, evidence is presented showing the preferential entrance of newly formed ribosomes into polysomes and the subsequent equilibration with the pool of single 74 s ribosomes.

2. Materials and Methods Oells The growth and harvesting of suspension cultures of HeLa cells have been adequately described previously (Eagle, 1959; Scherrer et al., 1963). All experiments were performed with exponentially growing cells at a concentration of 3 to 5 X 105/mI. Radioisotopes [2- 1 4C]uridine (30 fJ-c/fJ-mole) and [3H]uridine (2 to 3 mC/fJ-mole) were purchased from the New England Nuclear Corporation and were used as sterile solutions at 0·0006 to 0·01 msr, Techniques used in the assay of radioactivity have been described (Penman, Becker & Darnell, 1964). Oellfractionation Cytoplasmic extracts from HeLa cells were prepared in a hypotonic buffer (reticulocyte standard buffer or RSB, is 0·01 M-tris, pH 7,4, 0·01 M-KCI or NaCI,MgCI 2 as specified in each experiment) by Dounee homogenization and centrifugation of the nuclei (5 min at 1000 g) as has been previously described (Penman et al., 1963). Unless otherwise specified, the cytoplasmic extracts were treated with 0'5% DOC and 0'5% BRIJ·58, anonionic detergent (Atlas Chemical Industries, Inc., Wilmington, Del., U.S.A:). Isolation and sedimentation analysis oj RNA The release of RNA from cytoplasmic extracts or particles (Gilbert, 1963; Girard et at., 1964) was accomplished by the action of sodium dodecyl sulfate in buffer (0'5% sodium dodecyl sulfate, 0·01 M·tris (pH 7'4) and 0·1 M-NaCI). The RNA was precipitated from this buffer with two volumes of cold ethanol and then analyzed by zone sedimentation in various types of sucrose density-gradients (Britten & Roberts, 1960). Details of each experiment are given with the Figures in the text. Collection of gradients, ultraviolet monitoring and radioactivity assays on gradient fractions have been previously described (Penman et al., 1963; Penman et al., 1964).

3. Results Entry of ribosomal RNA into HeLa cell cytoplasm and demonstration of "native" 60 s and 45 s particles

In the initial description ofpolyribosomes (polysomes) in HeLa cells (Penman et al., 1963), it was shown that after a 35-minute exposure to 32p, more than 90% of the radioactive RNA associated with the polysomes differed from either ribosomal RNA or S-RNA in two ways: (1) its sedimentation profile was heterogeneous (6 to 25 s), and (2) its base composition resembled DNA. Since the polysomes were also shown to be the major site of cellular protein synthesis, the conclusion was made that the rapidly labeled polysomal RNA was messenger RNA (M-RNA). Of significance was the fact that although the vast majority of the total RNA in polysomes was R-RNA (16 sand 28 s RNA), the first radioactive RNA to reach the polysomes was M-RNA

RNA IN BeLa CELL CYTOPLASM

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plus a small amount oflabel in S-RNA. In order to characterize in more detail the flow of newly synthesized M-RNA and R-RNA into the cytoplasmic structures, cells which had been labeled for various periods of time were fractionated and the labeled RNA in polysomes was examined. Figure 1 shows the composite results of several experiments of this type. It can be seen that a 30-minute exposure to [3H]uridine (Fig. 1(a)) resulted in labeling only M-RNA in the polysomes, By 60 minutes (Fig. 1(b)), however, there was a mixture of ribosomal and M-RNA, and as the time of incorporation increased, the ribosomal RNA predominated to an increasing extent. The percentage of the total polysomallabel which is M-RNA at any time point represents an estimate arrived at by knowing the sedimentation profile of the sharply defined 16 sand 28 s RNA and subtracting the contribution of the R-RNA from the total. An experiment in which labeled M·RNA was mixed with increasing amounts oflabeled 16 s ribosomal RNA (Fig. 2) shows that a small amount of R-RNA (~ 10%) can be accurately detected in an excess of M-RNA. In practice, an estimate of the M·RNA content of polysomes can be made by simply drawing a base-line under the 16 sand 28 s peaks as shown by the lines bounding the shaded areas in Fig. 2. This method of determining the relative amounts of M-RNA and R-RNA in the polysomes is less accurate for R-RNA at early label times and for M-RNA at longer label times. Nevertheless, it is possible to see (1) the beginning of appearance of both 16 sand 28 s R-RNA in polysomes by this technique, and (2) the fact that, by 16 hours of exposure to label (Fig. 1), the proportion of polysomal RNA label in S-RNA and M-RNA together is 5% or less. In addition to the observations on the accumulation of labeled RNA in polysomes, the RNA from smaller structures (50 to 80 s) in the cytoplasmic extract was also examined. Fig. 1(a), (b) and (c) shows that by 30 minutes the smaller structures already contained labeled 16 s R-RNA, and by 60 minutes labeled 28 s RNA could be detected. The nature of the labeled RNA from the 50 to 80 s structures that does not sediment as 16 or 28 s RNA is unknown. It may of course represent M-RNA on the way into polysomes. In an earlier report (Darnell, Penman, Scherrer & Becker, 1963), the labeled R-RNA contained in 50 to 80 s structures was assumed to be in single ribosomes on the path. way to polysomes. Further examination, by the conventional sucrose density-gradient centrifugation for 90 to 120 minutes (Fig. 3), however, revealed no radioactive peak in the single ribosomes (74 s) even though labeling was extended to 120 minutes. There was, however, a large increase in total polysomallabel, which was shown in Fig. 1 to be largely R-RNA. In order to obtain better resolution of structures smaller than 74 s ribosomes, the sedimentation conditions were changed. Cytoplasmic extracts were centrifuged for 5 hours ("long spin") on a 5 to 20% sucrose density-gradient rather than the usual 90 to 120 minutes ("short spin"). A typical sucrose density-gradient analysis is given in Fig. 4 for a cytoplasmic extract which was divided into equal parts, one of which was subjected to a "long spin" and the other to a "short spin". The polyeomes are sedimented to the bottom of the centrifuge tube in the "long spin" (a), and can only be observed after a "short spin" (b). On the other hand, the single 74 s ribosomes are clearly separated from lighter material as well as polysomes only after a long spin. It has been found in many optical density tracings of this type that the amount of ultraviolet-absorbing material in polysomes is consistently five times greater than in single 74 s ribosomes. Cytoplasmic preparations from cells which had been labeled either 30 or 60 minutes were examined next by this "long spin" technique. Figure 5 shows that the structure

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FIG. 1. Labeled RNA from polyribosomes and smaller structures. Cytoplasmic extracts from growing HeLa cells which had been exposed to either [3H]_ or [HC]uridine were centrifuged (4°0, 120 min, 25,000 rev.fmin, SW35 rotor of Spinco model L) through 15 to 30% sucrose densitygradients in RSB. The polysome reg ion was collected, RNA released by 0'5% sodium dodecyl sulfate in presence of non-radioactive carrier cytoplasm, precipitated with 2 vol, of - 15° ethanol, redissolved in sodium dodecyl sulfate buffer (0'5% sodium dodecyl sulfate, 0·01 M-tris (pH 7,4) 0·1 M-NaCl) and examined on a 15 to 30% sucrose density-gradient in sodium dodecyl sulfate buffer (20°C, 15 hr, 23,000 re v.fmin, SW25 rotor). The analysis of radioactivity ( - 0 - 0 - ) and ultraviolet absorbing material (----) of this second gradient from the various samples is given: (a), 30 min; (b), 60 min; (c), 100 min; (d), 200 min; (e), 16 hr. In addition to the polysomes of all the samples, the 50 to 80 s region of the initial sucrose gradient was also collected and RNA examined from three samples (a'), 30 min ; (b'), 60 m in; (c'), 120 min. The labeling conditions for the variou s cul tures were as follows : (a), 150 ml. culture, [3H]uridine, 500 P.c, 0·16 p.mole; (b), same as (a); (e) and (d), 100 ml, culture, [HC]uridine, 5 P.c, 0·16 p.mole; (e), 100 ml, culture, [HC} uridine, 1 P.c, 1 p.mole.

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FIG. 2. Resolution of mixture of 16 s R-RNA with M-RNA. M-RNA uncontaminated with 16 s R-RNA was prepared from polysomes of briefly labeled cells (see Fig. l(a» and mixed with three different amounts of 16 s R-RNA obtained from ribosomes of cells labeled for a generation (see Fig.l(e». The mixtures were of the following proportions: (a), 12% R-RNA; (b), 25% R-RNA; (c), 60% R-RNA. The mixtures were then analyzed by sucrose density-gradient analysis as in Fig. 1 and the amount of 16 s R-RNA discernible in each sample estimated by summing the counts above the lines drawn under the 16 s peaks. The total recovered counts as 16 s and as M-RNA are given in the Figures. The recoveries of label approached 100% in each case, and the estimates show that as low an amount of 16 s RNA as 1/10 of the total can be accurately estimated. 0.0.

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FIG. 3. 450 ml. of growing cells were labeled with I me [3H]uridine, 0'3 f'mole, for 60 (-E9-E9-), 90 (-0-0-) and 120 (-e-e-) min. Cytoplasmic extracts were prepared in RSB, 0·005 MMg2+, and analyzed by sucrose density-gradient analysis in 5 to 20% sucrose RSB, 0·005 M-Mg2+, (25,000 rev./min, 4°C, 120 min). Only the optical density tracing for the 120·min sample is given by the solid black line. About two-thirds of the polysomes have already reached the bottom of the centrifuge tube in these gradients.

192

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FIG. 4. Comparison of cytoplasmic extract analyzed by "short spin" and "long spin". Cytoplasmic extract from 200 ml. of culture was divided. One-half (b) was examined for ultraviolet. absorbing material on a 15 to 30% sucrose density-gradient (in RSB, 0·005 M.Mg 2+ ) by a 90 min centrifugation at 25,000 rev.fmin, 4°C ("short spin"). The other half (a) was sedimented for 5 hr at 25,000 rev.fmin ("long spin"). The total optical density under the profile of the polysomes is approximately 5 times that of the single ribosomes.

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FIG. 5. A growing culture of HeLa cells was labeled with [3H]uridine and samples fractionated at 30 min (a) or 60 min (b). The cytoplasmic extracts were examined by sucrose density-gradient centrifugation (Mg 2 + concn. 0-003 M, centrifugation as in Fig. 2) for radioactivity ( - 0 - 0 - ) and ultraviolet-absorbancy (----). The RNA from the 45 s particles (inset, (a» was examined as in Fig. 1. A sample of labeled cells identical to (a) was fractionated as described in Materials and Methods, except that just prior to Dounce homogenization at 4°C concentrated sucrose was added to 0·25 M (isotonic). The optical density and radioactivity profile was indistinguishable from (a).

RNA IN HeLa CELL CYTOPLASAI

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containing the majority of the newly formed 16 s R-RNA after a 30-minute label has an S value of 45 s approximated by comparison with the 74 s single ribosomes (Martin & Ames, 1961). Cytoplasmic extracts from an identical sample of cells to those shown in Fig. 5(a) were prepared under isotonic conditions and the number of 45 s particles was unaffected. This suggests, but does not prove, the hypothesis that 45 s particles existed in the cytoplasm before cell fractionation. With longer exposure to radioisotope (60 to 70 minutes) more radioactive material accumulated in the 45 s particles, and label then began to appear as a peak in the 60 s region also. At least 80% of the radioactivity in the 45 s particle was 16 s R-RNA (Fig. 5), while the 60 s structures appeared to have both 16 and 28 s RNA (not shown in Fig. 5; see Fig. 6). In order to determine whether the 16 s RNA found in the 60 s region was a contamination from the 45 s particles, conditions were sought which would allow the separation of 60 s structures free of 45 s native particles. It was found that ifthe detergent BRIJ-58

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FIG. 6. Effect of detergents on precipitation of sub-ribosomal particles. Cells which had been labeled for 30 min with [3H]uridine were fractionated in RSB, 0·003 M_Mg 2 + , without the addition of either DOC (No, desoxycholate, Mann) or BRIJ. One sample, (a), was immediately layered onto a 5 to 20% sucrose density-gradient for analysis. DOC, 0'5% final concentration, and BRIJ-58, 0'5% final concentration, were added to (b) whereas DOC alone was added to (c). Note (c) that not only did the DOC alone cause the precipitation of the labeled 45 s particles but also the 60 sand 45 s structures which were observed in (a) and (b) by ultraviolet absorption. In part (d), an extract of cells labeled for 70 min was examined with DOC but without BRIJ treatment. The 60 s labeled structures (F) and material sedimenting under the 74 s peak (E) were taken for analysis of RNA content by sodium dodecyl sulfate extraction as in Fig. 1.

194

M. GIRARD, H. LATHAM, S. PENMAN AND J. E. DARNELL

was omitted during the preparation of a cytoplasmic extract, but deoxycholate was added as usual, the 45 s native particles disappeared in sucrose density-gradient analysis, but most of the 60 s native particles remained. Figure 6 demonstrates this phenomenon, which is due to precipitation of the 45 s structures in the absence of BRIJ and the presence of DOC. The 45 s which disappears from a sample treated with DOC alone does not appear in polysomes (see, for example, Fig. l(a)), but can be recovered from the bottom of the centrifuge tube. After brief sedimentation, it should also be noted that the small amount of ultraviolet absorbing material which forms discrete peaks at 60 sand 45 s in Fig. 6(a) also largely disappears from the gradient when only DOC is used. This material represents dissociated polysomes and ribosomes observed at 0·0015 or 0·003 M_Mg 2 + , and is discussed in detail later in this paper. The precipitation phenomenon discussed above always occurred at 0·0015 M_Mg 2 + but was observed irregularly at 0·003 M_Mg 2 +. In some experiments at 0·003 M_Mg 2 + the 60 s particles were also precipitated to a large extent. Presumably uncontaminated native particles were examined by this technique for labeled RNA content after a 70-minute label. Figure 6(d) and (f) shows that the 60 s 'structure still contained both 16 and 28 s. Moreover, the majority of the label was 16 s, arguing against the position that the 60 s peak was truly composed of a structure containing only 28 s contaminated by 45 s native particles. In addition, structures from the 74 s region (Fig. 6(e)) also had the same ratio (about 2 to 1) of 16 to 28 s RNA which is characteristic for polysomal ribosomes after this length oflabel (Fig. 1). One final observation against any significant forward spreading of 45 s structures into the 60 s region is given in Fig. 9. There it is shown that 45 s structures which are re-run on sucrose density-gradients do not spread into the 60 s region to any appreciable extent. The 60 s structures would therefore seem to be either (1) particles containing both types of RNA, or (2) a 60 s particle containing only 28 s RNA plus a very specific aggregate of 45 s particles containing only 16 s RNA which sediments at 60 s. In order to decide unambiguously about the nature of the 60 s native particle, it will be necessary to separate these structures on the basis of some physico-chemical characteristic other than size and to determine if the 16 s RNA from the 60 s region stays with the 28 s RNA. Effect of magnesium ion concentration on HeIa cell ribosomes and polysomes

Since whole ribosomes (70 to 80 s) from a variety of sources including HeLa cells (Ts'o, Bonner & Vinograd, 1958; 'I'issieres, Watson, Schlessinger & Hollingworth, 1959; Lederberg & Mitchison, 1962; Attardi & Smith, 1962) will dissociate into two unequal subunits (50 to 60 sand 30 to 40 s) at low concentrations of Mg 2 + , a comparison of "native" and derived subribosomal particles was made. The stability of ribosomes and polysomes in cytoplasmic extracts of HeLa cells prepared at various Mg 2 + ion concentrations in the standard buffer (RSB, see Materials and Methods) was tested first. It was found that the yield of cytoplasmic polyribosomes as measured by ultraviolet absorption was not measurably changed in Mg 2 + concentration of 0·0015 M (a standard concentration which has been used in much of the work with animal cell polyribosomes) down to 0·00015 M_Mg 2 + . Concentrations up to 0·005 Min RSB also do not affect the yield of polysomes; but above 0·005 Mprecipitation begins to occur. A more sensitive test for dissociation was to examine single ribosomes prepared at 0·005 M-Mg 2 + from a culture which had incorporated [3H]uridine for one generation.

RNA IN HeLa CELL CYTOPLASM

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FIG . 7. Effect of Mg 2 + concentration on HeLa cell ribosomes. HC·lab eled sin gle ribosomes (74 s) were prepared by sucrose d en sity-gradient isolation at 0·005 1I1.M g 2 + fr om cells exposed to [HC} uridine for two gen eration s an d a dde d back to the cytopla smic ex t ract of the equiv al en t o f 2 X 10 7 unlabeled H eLa cells at various Mg2 + conc en t rati on s ; (a), 0·005111 ; (b) , 0·001 5 M; (c), 0 ·00 015111. The mixtures wer e then centrifuged again through suc rose densit y -gradients t o test the dissociation of 74 s ribosomes t o smaller structures . Cen t rifugat ion cond it ions : 5 hr at 4°C, 25,0 00 r ev .fmin, 5 to 20 % sucrose in RSB conta ining a ppropriate Mg2+ co ncentrations. I ndicat ed frac t ions (1, 2, 3) were taken for determination of RNA content as d escribed in Fig. 1.

Figure 7 shows some degradation into two smaller st ruc tures at 0·0015 M-:NIg2 + and additional dissociation at 0'00015 M . The extent of dissociation of the total cytoplasmic ribosomes (polysomes plus ribosomes) as a function of Mg 2 + concentration was determined. About 85 to 90% of the ribo somes and polysomes ar e sta ble at Mg 2 + concentrations greater than 0·0015 M and 90 to 95% above 0·003 M . The relative stability of single ribosomes versWl polysomes was not determined directly. Figure 7 shows that the ribosomal dissociation products (low Mg2 + particles) sediment (in the presence of cytoplasmic 'extract ) with S value s of 60 and 45 s , just as the "native" particles do (Figs 5 and 6); although the native 60 s may contain both 28 and 16 s RNA, while the low Mg 2 + concentration 60 s particle contains only 28 s RNA. Attardi & Smith (1962) had, however, reported lower S values (50 sand 30 s) for sub-ribosomal particles derived from purified ribosomes by more extensive removal of Mg2 + . A comparison was made, therefore, between low Mg 2 + concentration par. ticles and particles derived from ribosomes and poly somes by EDTA treatment (EDTA particles). The EDTA-treated ribosomes and poly somes dissociated into two structures, which were purified by sucrose density.gradi ent centrifugation (Fig. 8). When the purified EDTA-derivedparticles were compared at 0·0015 M_Mg 2 + to 74 s ribosomes or to the low.Mg2 + concentration particles, they were found to sediment at 50 and 30 S, containing only 28 sand 16 S RNA respectively. If the EDTA par ticles were placed back in a cyt oplasmic extract at 0·0015 M-Mg2 + , the majority of 50 s and 30 s particles now sedimented at 60 sand 45 s respectively (F ig. 8(c) and (dj ). Although the cause of this shift from 50 to 60 s and from 30 to 45 s in sedimentation coefficient is unknown, it appears to require exposure to cyt oplasmic extract and is not mediated merely by 0·0015 M_Mg2 +.

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FIG. 8. HeLa cell ribosomes in EDTA. Part (a): A cytoplasmic extract from about 2 X 107 HeLa cells which had been labeled for 16 hr with [14C]uridine was prepared in RSB without Mg 2 + • After separation from the nuclei, the extract was made 0·001 M in EDTA, layered on 15 to 30% sucrose density-gradient in the same EDTAcontaining buffer and centrifuged for 15 hr, 20,000 rev.fmin at 4°0. Samples were taken from the two ultraviolet-absorbing peaks for determination of the type of radioactive RNA in each peak after the addition of cold cytoplasmic RNA «a), inset). Part (b): Approximately 0·05 m!' of each of the two peaks seen in (a) were added back to 1 rnl, RSB containing 0·0015 M-Mg2+ plus 0·05 ml. of 0·003 M_Mg 2 + to provide excess Mg2+ for the small amount of EDTA. The mixture was then layered on a 15 to 30% sucrose density-gradient containing RSB and 0·0015 M_Mg2+ and centrifuged for a "long spin"; and radioactivity determined (--0--0--). A separate sample containing cytoplasmic extract in RSB at 0·0015 M.Mg 2 + was centrifuged at the same time to provide an optical density marker (--). Part (c): A sample of the larger radioactive EDTA subunit (50 s) added to carrier cytoplasmic extract (0-0015 M_Mg2+) rather than to RSB, and analyzed as in (b). Part (d): A sample of the smaller radioactive EDTA subunit was added to carrier cytoplasmic extract and analyzed as in (b).

RNA IN HeLa CELL CYTOPLASM

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One further observation was that when the rapidly labeled 60 and 45 s native particles were directly compared to the 50 and 30 s EDTA particles in the absence of cytoplasmic extract, the difference in sedimentation pattern was maintained (Fig. 9). Therefore, if the 16 s radioactivity shown to be present in 60 s native particles is due to aggregation of 45 s particles, then the aggregation is stable when the particles are very dilute.

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Relationships between free single ribosomes and polysomes

The next series of experiments was designed (1) to quantitate the appearance of first, the 16 s ribosomal RNA, and then the 28 s ribosomal RNA, in native particles prior to ribosomes and polysomes; and (2) to observe the appearance of R·RNA in single ribosomes compared to polysomes after longer label times. Table 1 gives a summary of several experiments in which cells were labeled for 30, 40, 45 and 50 minutes and fractionated so that the total labeled ribosomal RNA could be estimated in (1) polysomes, (2) under the 74 s optical density peak, (3) 60 s particles, and (4) 45 s particles. It can be seen that the 16 s R·RNA first appears in 45 s particles, followed by 60 and ~ 74 s structures. It should be emphasized that the material sedimenting in the 74 s region does not follow the optical density in that region at early label times (see Figs 3,5 and 6). With slightly longer label times (50 minutes) the 28 s makes its appearance first in the 60 s and then ~ 74 s structures before it is found in poly. somes. The native smaller structures are, therefore, precursors to finished ribosomes, as judged by the fact that they are the initial sites of appearance of R-RNA. It has not 14

M. GIRARD, H. LATHAM, S. PENMAN AND J. E. DARNELL

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1 Distribution of ribosomal RNA in various cytoplasmic structures TABLE

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28 s RNA

608

,...., 748

1000 1100 3000 3000

250 300 360 400 500

Poly. somes lOOt 150t 1300 1100 5000 12,500

458

608

74 s

Poly. somes

None at this time

< <

100 100

750§ 700

50 200

< < <

100 100 100

Results obtained from three experiments of fractionation of cytoplasmic extracts into particulate fractions (45, 60, 73 8 on "long spins" and polysomes by "short spins") performed as described for Fig. 4 and subsequent examination of RNA as described in Fig. 1. Note that the 748 column indicates that a sample was removed from the 74 s region, but that radioactivity did not correspond to optical density in this region after brief labels (see text and Fig. 5). t First appearance of 16 s RNA. t Apparent absence of 16 s or 28 8 counts; figure given is limit of number of counts that should have been detected. § First appearance of 28 8 RNA.

been possible to carry out effective chase experiments to provide unequivocal evidence that material in native particles ends up in finished ribosomes for two reasons. First, the cessation of uptake of uridine after a chase by non-radioactive nucleotides is not effective for approximately 20 minutes. Second, there probably is intranuclear turnover of some of the rapidly incorporated radioactive material which causes label to continue to migrate from the nucleus for a relatively long period of time (Harris, 1963; Scherrer et al., 1963). The next question considered was the relationship between single 74 s ribosomes and polysomes. Although radioactive R-RNA can be found under the 74 s optical density peak after brief label periods, there is not a correspondence between radioactivity and optical density in the 74 s region. Moreover, the experiment illustrated by Fig. 10 shows that, after a continuous 70·minute label, the specific activity of R-RNA in polysomes is three times higher than R-RNA in single ribosomes which have been purified by two isolations in sucrose density-gradients. On the other hand, the R· RNA's of polysomes and free single 74 s ribosomes at 3, 3·75 and 4·5 hours after labeling (Fig. 11) are equal in specificactivity, although a threefold increase has occurred in both fractions. In order to understand these two experiments, it is necessary to recall (Fig. 3) that labeled R·RNA is entering the cytoplasm very rapidly after about 60 minutes. For example, the total R-RNA in polysomes (corrected by subtraction ofM.RNA) at 120 minutes is about 6 to 8 times greater than at 60 minutes (Fig. 3). The difference is, of course, greater still between 70 minutes, the label time used in Fig. 10, and 3 to 4·5 hours, the label time used in Fig. 11. Thus the lag in equilibration between poly. somes and single ribosomes which is observed with the shorter labels (Figs 3, 5, 6 and 10), but not the 3 to 4·5 hour label (Fig. 11) is interpreted as follows. A newly synthesized ribosome goes straight into polysomes without passing into the total free

RNA IN HeLa CELL CYTOPL A SM

(0)

cts /min 00. (o--~)

(-)

5000

1·0

199

(b)


01

o

80 %R-RNA 40 % R- RNA

10

20

30

Fraction No.

FIG. 10. Cells wh ich had been la beled for 70 min were fract ion at ed , an d half t he cy to plasmic extract was spun for 2·5 hr on a 5 to 20 % suc rose densit y -gr ad ient (a), while the ot her half was spun for the sa me t ime on a 15 to 30% gradient (b ). N ote that t he different concentrations of sucro se h as allowed a large fr action of the polyso mes in (a) t o m ove to the bottom of t he t ube. Polysomes were pooled from (b), RNA ext ract ed and analyze d for R-RNA content as in Fi g. 1 without a ny carrier RNA b ein g added . The spec ific activit y of R-RNA in p olyso mes was 4400 cts/ m in /o .D. unit. The leading edge of t he single ribosome p ea k was t aken from sample (a ) an d re run (15 hr, 20,000 re v ./min, 4°0 on a 15 to 30% gradient) to sepa rate sing le ribosom es clearly. T he sp ecific activity of the R-RNA in a sin gle ribosome was 1380 ct s/ m in/ o.D. unit .

15,000

•

-c z cr: I cr:

o

'1510,000 ci

o L

OJ

a.. c:

J.

...,u>

5000

U

4·5

3 (hr)

FIG. 11. Cultures labeled for 3, 3·75 and 4·5 hr were fract ionated a nd analyze d as in F ig. 10, and tho specific activities of R-RNA in free single ribosom es (74 O.D. peak ) and polysomes deter . mined. (0, polysomes; .,74 s ribosomes).

200

M. GIRARD, H. LATHAM, S. PENMAN AND .J. E. DARNELL

74 s pool. After a brief time, the new ribosome does appear in the total free 74 spool. By three hours, the labeled free ribosomes are in excess over any newly labeled ribosomes which have not yet been through an initial polysome association. After three hours, the further increase in specific activity of polysomal R-RNA is matched by the increasing radioactivity in R-RNA from single 74 s ribosomes, indicating a rapid equilibrium between most of the single ribosomes and most of the polysomes. These observations are strong evidence against a necessity for the ribosomes on a new polysome to continue synthesizing from one M-RNA molecule until it is destroyed. The 3 to 4-hour half-life ofM-RNA (Penman et al., 1963) would allow the release only every 45 minutes of a number of single ribosomes equivalent to the free 74 spool (onesixth of total ribosomes). Equilibration of polysomal R-RNA with R-RNA in unattached ribosomes would therefore take at least 45 minutes, even if the specific activity of polysomes did not increase in the observed period. Since no appreciable lag is detected (Fig. 11) between the total polysomes and total ribosomes, it is concluded that free exchange occurs between the two. It is impossible to say whether an exchange occurs with each peptide chain completed. The cause of the initial delay of polysome and ribosome equilibration might be due either to slowness of initial function of a newly formed ribosome, or an enhanced chance for involvement in polysomes by a new ribosome due, for example, to its proximity to the nucleus.

4. Discussion Ribosomal RNA is found in HeLa cell cytoplasm in subribosomal particles before it can be identified in whole ribosomes or polysomes. One of these particles (the 45 s) appears to be identical to a ribosomal subunit obtainable from completed ribosomes at low Mg 2 + concentrations. This 45 s subunit may be more compact (thus faster sedimenting) than the smaller ribosomal subunit derived by EDTA treatment. A large precursor (60 s) to completed ribosomes is also identifiable. It is not clear whether this structure is identical to the larger, low Mg2 + concentration subunit from whole ribosomes, or whether it represents an almost finished ribosome with both 28 and 16 s ribosomal RNA. It has been assumed on the basis of electron microscopic and radioautographic evidence that ribosomes are finally assembled, if not indeed entirely synthesized, within the nucleolus, an organelle within the nucleus (Swift, 1959; Marinozzi, 1962; Perry, 1962; Edstrom, 1960). Recent work utilizing gradient centrifugation of the nucleolar contents of plant cells has established that subribosomal particles can indeed be found in the nucleolus (Birnstiel, Chipchase & Hyde, 1963). In the present study, therefore, it was somewhat surprising to find precursors to whole ribosomes in the "cytoplasmic" extracts of HeLa cells. These structures were present in cellular extracts (prepared under either hypotonic or isotonic conditions) where no more than 3% of the cell DNA was found in the "cytoplasmic" fraction. These findings could, of course, be explained by assuming that ribosomal precursors which are finished, or almost finished, structures are released from the nucleolus when the cell is broken, or alternatively it could be that the final step of ribosome formation occurs in the cytoplasm. One final point with respect to the physiological assembly of ribosomes requires comment. The appearance of 16 s RNA prior to 28 s RNA in the cytoplasm is strikingly evident in several experiments (Fig. 1, Table 1). This asynchrony of arrival in

RN A IN HeLa CELL CYTOPLASM

201

the cytoplasm of the two types of R-RNA exists, although both molecules are metabolically stable and are found in the cell in equimolar amounts. In the growing cell, therefore, a molecule of 28 s R·RNA must enter the cytoplasm with each 16 s if the R-RNA is truly metabolically stable. Perhaps the most satisfactory explanation which accounts for these findings is that the 28 s-containing portion of the ribosome is more complicated and takes longer to complete than the 16 s-containing portion. Thus, although a labeled molecule of 16 and 28 s RNA might enter the assembly process simultaneously, the 16 s would emerge first, accompanied by an unlabeled 28 s which had been synthesized previously. One of the authors (M. G.) is a Fellow of Cornite de Biologie Moleculaire, Delegation General S la Recherche Scientifique et Technique, Paris, France. Another of the authors (S. P.) is a Special Fellow, U.S. Public Health Service. This work was supported by grant C-5789 from the U.S. Public Health Service and grant GB-513 from the National Science Foundation. REFERENCES Attardi, G. & Smith, J. (1962). Cold Spr. Harb. Symp. Quant. Biol. 27, 271. Birnstiel, M. L., Chipchase, M. & Hyde, B. B. (1963). Biochim. biophys. Acta, 76, 454. Britten, R. J. & Roberts, R. B. (1960). Science, 131, 32. Darnell, J. E., Penman, S., Scherrer, K. & Becker, Y. (1963). Cold Spr. Harb. Symp. Quant. Biol. 28, 211. Eagle, H. (1959). Science, 130, 432. Edstrom, J. E. (1960). J. Biophys. Biochem, Cytol. 8, 47. Gierer, A. (1963). J. Mol. Biol. 6, 148. Gilbert, W. (1963). J. Mol. Biol. 6, 389. Girard, M., Penman, S. & Darnell, J. E. (1964). Proc. Nat. Acad. e«, Wash. 51, 205. Harris, H. (1963). Nature, 198, 184. Harris, H., Fisher, H. W., Rodgers, A., Spencer, T. & Watts, J. W. (1963). Proc. Roy. Soc. A, 157, 177. Lederberg, S. & Mitchison, J. M. (1962). Biochim. biophys. Acta, 55, 104. Marinozzi, V. (1962). Proc, 5th Inst, Conf. Electron Micros., p. 200. New York: Academic Press. Martin, R. G. & Ames, B. N. (1961). J. Biol. Chem, 236, 1372. Penman, S., Becker, Y. & Darnell, J. E. (1964). J. Mol. Biol. 8, 541. Penman, S., Scherrer, K., Becker, Y., & Darnell, J. E. (1963). Proc. Nat. AcadSci., Wash. 49,654. Perry, R. P. (1962). Proc, Nat. Acad. Sci., Wash. 48, 2179. Scherrer, K. & Darnell, J. E. (1962). Biochem. Biophys. Res. Comm. 7,486. Scherrer, K., Latham, H. & Darnell, J. E. (1963). Proc. Nat. Acad. Sci., Wash. 49, 240. Swift, H. (1959). Brookhaven Symp. BioI. 12, 134. Tissieres, A., Watson, J. D., Schlessinger, D. & Hollingworth, B. R. (1959). J. Mol. Biol. 1,221. Ts'o, P. O. P., Bonner, J. & Vinograd, J. (1958). Biochim. biophys. Acta, 30, 570. Warner, J. R., Knopf, P. M. & Rich, A. (1963). Proc. Nat. Acad. Sci., Wash. 49, 122. Wettstein, F. 0 .• Staehelin, T. & Noll, H. (1963). Nature, 197, 430.