J. Mol. Biol. (1969) 39, 545-550

Molecular Weights of Some HeLa Ribosomal RNA’s

ED H. McCoNKEYt AND JOHNS W. HOPKINSIS

The Biological Laboratories, Harvard University Cambridge, Mass. U.S.A.

(Received 15 July 1968)

The molecular weights of HeLa ribosomal RNA’s, determined by sedimentation equilibrium at pH 7.0, were found to be: 45 s RNA, 4 to 4.5 x lo6 daltons; 32 s RNA, 2.4 x lo6 daltons; 28 s RNA, 1.9 x lo6 daltons; 18 s RNA, 0.71 x lo6 daltons. These values imply a non-conservative transition from the 45 s precursor to the mature 18 and 28 s ribosomal RNA%.

1. Introduction In 1962 Scherrer & Darnell described two rapidly labeled RNA’s with high sedi- mentation coefficients (now called 45 and 32 S) from HeLa cells. Subsequent work in several laboratories has strongly implicated these RNA’s as precursors of the 28 and 18 s mature ribosomal RNA’s (reviewed by Perry, 1967; Birnstiel, 1967). The exact relationship of the precursor molecules to the mature rRNA’s and the details of the transition from one stage to another remain unknown.

In a recent communication, Weinberg, Loening, Willems & Penman (1967) have described additional steps in the maturation of rRNA. The relationships proposed by them are as follows:

--+20s?+18s

L 32 s -28 s

On the basis of measurements of the relative extent of methylation of the 45, 32, 28 and 18 s RNA’s, Weinberg et al. (1967) have proposed that the transition from 45 to 18 and 28 s molecules is non-conservative. Their hypothesis has been strengthened further by the finding that the base composition of 45 s RNA is not equal to the weighted average base composition of 18 plus 28 s RNA (Willems, Wagner, Laing & Penman, 1968). Here we present molecular weight data which support their conclusion. These data have already been cited by Perry (1967) and by Willems et al. (1968).

2. Materials and Methods

We have measured molecular weights of RNA’s by the high-speed sedimentation equi- librium technique of Yphantis (1964), which is capable of yielding moderately accurate results with only a few micrograms of sample. A model E ultracentrifuge equipped with interference optics was used. Details of the conditions of centrifugation are given in the

-iPresent address: Department of Molecular, Cellular and Developmental Biology, University of’ Colorado, Boulder, Colorado 80302, U.S.:\.

SPresnnt address: Department of Biology, \Vnshington University, St,. Louis, MO., 63130, U.S.A. Vi45

846 E. H. McCONKEY AND J. W. HOPKINS

legends to the Figures. Data were recorded as microns of fringe displacement from the zero oonoentration level, which is a measure of solute concentration, at multiple points along the radius of the cell. Apparent molecular weights were then calculated from the formula:

M = d(ln 4 . 2RT . W) wa(l-i$)

The first term on the right aide of the equation was obtained directly from the slope of the plot of fringe displacement wereus rz. The symbols in the second term have their conven- tional meanings (R, gas constant; T, absolute temperature; W, angular acceleration; 8, partial specific volume; p, density).

The 18 and 28 s RNA’s were obtained from extracts of whole cells. Extractions were done at 4”C, using phenol-O+“/o sodium dodecyl sulfate in O-01 BI sodium acetate, pH 6.1. The 32 and 46 s RNA’s were obtained from nucleoli Golated essentially ss described by Penman, Smith & Holtzman (1966). Instead of warming the nuclear homogenate during DNase treatment, we kept it in an ice bath for 15 min, and we used two treatments with DNase. Nucleolar RNA’s were purified according to the hot chloroform technique of Penman (1966). Eaoh size class of RNA was separated by oentrifugation through two sucrose gradients sequentially. The preparations used for equilibrium sedimentation were apparently monodisperse, as judged from the symmetry of the peaks on the final sucrose gradients.

3. Results (1) The 18 s RNA has an apparent molecular weight of 0.71 x lo6 daltons (Fig. 1).

This value is in satisfactory agreement with other reported determinations of the molecular weight of mammalian 18 s rRNA. Petermann t Pavlovec (1966) used sedimentation velocity and viscosity to obtain a figure of 0.67 x lo6 daltons for the same RNA from Jensen sarcoma.

(2) The 28 s RNA has an apparent molecular weight of 1.9 x 10s daltons. This figure is unusually high. Most values in the literature are in the range of l-6 to 1-7x lo6 daltons (Gierer, 1958; Hamilton, 1967; Petermann & Pavlovec, 1966). Nevertheless, we believe our measurement to be correct. In the 6rst place, our data show no

500

5

-2 2 $j 250 3 .

0 0) P .-

Lk

E 100 37,5 37.8

f* (cm*)

FILL 1. 18 s RNA after attainment of sedimentation equilibrium. Sample oontained 10 pg of RNA in 0.1 ml. of 0.14 ~-N&I-0*01 M-EDTA, pH 7.0. Centrifugstion

wt~ done in the Yphantis 6-charmel centerpiece, using the AN-J rotor at 6569 rev./ruin, at 6’C. The graph presents date cmloulated from a photograph taken after 48 hr of centrifugation. In&x- ferenoe optics were used. M,,, is 0.71 x lo6 daltons.

HELA RNA 547

evidence of aggregation (Pig. 2(b)), which would have been clearly indicated by an upward curvature of the plots of fringe displacement versus r2. Second, this RNA was prepared by cold phenol extraction of whole cells. On sucrose gradients there was no indication of degradation. Third, it has been shown recently (Pene, Knight & Darnell, 1968) that a small 7 s piece of RNA can easily be split away from HeLa 28 s

rRNA during purification. Many of the “28 S” RNA’s previously studied may have lacked this 7 s piece. The molecular weight of the 7 s piece has been estimated to be approximately 40,000 daltons.

I-~ (cm2) r2 (cm*)

(a) (b)

FIG. 2. 28 s RNA after attainment of sedimentation equilibrium. (8) Sample contained 20 pg of RNA in 0.1 ml. of 0.14 M-NaCl-0.01 M-EDTA (pH ‘7.0)-l%

HCHO. (b) Semple contclined 20 pg of RNA in 0.1 ml. of 0.14 M-NaCl-0.01 M-EDTA, pH 7.0. Both

samples were centrifuged simultaneously at 3848 rev./min. Other conditions 8s in Fig. 1. The graphs present data calcul8ted from a photograph taken after 74 hr of centrifugetion. M,,, is 1.68 x lo6 d8ltons (8) and 1.88 x lo6 daltons (b).

It should be noted also that our molecular weight determinations refer to the sodium salt of RNA at pH 7.0, whereas those of Petermann & Pavlovec refer to the nucleic acid with non-ionized phosphate groups at pH 4.6. The molecular weight of the sodium salt of RNA is approximately 107% that of the non-ionized form of the molecule. Our measurements therefore imply a molecular weight of 1.77 x IO6 daltons and 0.67 x lo6 daltons for the acidic forms of 28 and 18 s RNA’s, respectively.

(3) In order to obtain measurements of the molecular weight of the 32 and 45 s RNA’s, we were forced to modify the buffer. In the 0.14 M-NaCl-0~01 M-EDTA, pH 7-O buffer used for 18 and 28 s RNA’s, we observed extensive aggregation of the 32 and 45 s RNA’s. This difficulty was overcome by the addition of 1% HCHO to the buffer. Figure 3(a) and (b) show no indication of the presence of aggregates when the 45 and 32 s RNA’s were centrifuged to equilibrium in the HCHO buffer.

The effect of 1% HCHO on the molecular weight determinations is difficult to predict. In practice, we find that the apparent molecular weight of 28 s RNA is approximately 10% less in formaldehyde buffer (1.7 x lo8 daltons) than in the buffer

E. H. McCONKEY AND J. W. HOPKINS

.

I I 44.0 44.5

r-2’ km3

(a)

r* (cm*)

(b)

FIG. 3. 45 s RNA (a) and 32 s RNA (b) after attainment of sedimentation equilibrium. Each sample contained 10 pg of RNA in 0.1 ml. of 0.14 M-NaGI-0.01 m-EDTA (pH 7.0)-l% HCHO. The graphs present d8t8 calculated from a photograph taken after 67 hr of centrifugation at 3189 rev./min. Other conditions as in Fig. 1. M,,, is 3.82 x lo8 daltons (a) and 2.11 x lo8 daltons (b) before adjustment. See text.

This speed (3189 rev./min) is about 30% higher than the optimum speed for a molecule the size of 45 s RNA (Yphentis, 1964). It was used to avoid precession of the rotor at lower speeds, and to allow comparison of the 45 and 32 s RNA’s in the same run. Precession was not noticeable at this speed, or at the higher speeds used for the smaller RNA’s.

without formaldehyde (l-9 x lo6 daltons). We have therefore estimated molecular weights for the 45 and 32 s RNA’s as follows:

mol. wt. 45 s (or 32 s) = mol. wt. 45 s (or 32 S) in HCHO mol. wt. 28 s not in HCHO

mol. wt. 28 s in HCHO

The apparent molecular weight of the 32 s RNA is calculated to be 2-4 x lo6 daltons by this method.

We should also like to point out that Figure 3(b) shows no indication of the presence of molecules smaller than the 32 s RNA. We infer that the 32 s RNA was effectively separated from 28 s RNA by our preparative procedure. The critical step appears to be a second treatment of the nucleoli with DNase, which is not called for by the procedure of Penman et al. (1966). Strict application of the latter procedure always yields a significant amount of 28 s RNA, as emphasized by Weinberg et al. (1967). Whether the 28 s RNA in question actually was present in the nucleolus in situ, or whether it represents ribosomal precursors trapped by nucleolus-associated chromatin, we cannot say.

(4) The apparent molecular weight of 45 s RNA is at least 4 x lo6 daltons, and probably 4.5 x lo6 daltons. We cannot be more precise, because there is evidence for the presence of some lower molecular weight material in our 45 s RNA’s at equilibrium (Fig. 3(a)). Weinberg et al. (1967) have recently demonstrated by acrylamide gel electrophoresis that the nucleolus also contains 41 s and 36 s RNA’s. Our 45 s RNA may have contained some 41 s RNA, or the 45 s RNA may have been slightly degraded.

HELA RNA 540

4. Discussion (1) The absolute magnitude of the molecular weights reported here is uncertain.

For that reason, we have used the term apparent molecular weight. The uncertainty exists because the usual equation for sedimentation equilibrium is not strictly applicable to charged polyelectrolytes. A more precise estimate of the actual weight- average molecular weight of these RNA’s could be obtained if the procedure of Casassa & Eisenberg (1964) were used to calculate 8, the partial specific volume. In this study, we have used a value T?, 053 ml./g, reported by Stanley & Bock (1965), and by Petermann & Pavlovec (1966). Although all of the molecular weights calculated with this partial specific volume may be wrong by an unknown small amount, the relative molecular weights will not change. This is essential to the interpretation that follows.

(2) Our molecular weight measurements agree rather well with the molecular weights that can be predicted from the sedimentation coefficients of the RNA’s (Spirin, 1961; Gierer, 1958).

(3) The 32 s is believed to be a precursor of the 28 s RNA, but not of the 18 s RNA. This conclusion rests principally on the fact that radioactive precursors appear simultaneously in the 32 and 18 s RNA’s (Penman, 1966; Zimmerman & Holler, 1967). The data presented here show that the 32 s RNA is too small to be a precursor of both 18 and 28 s RNA’s, but too large to be considered a mere configurational variant of 28 s RNA, which could conceivably result from a difference of a few methyl groups, for example. In fact, the molecular weight of 32 s RNA is approx- imately 27% greater than that of 28 s RNA.

We conclude that a large fragment is removed from 32 s RNA when it is converted to 28 s RNA. We do not know the identity or fate of this fragment, but we should like to point out that it is too large to be nothing more than a precursor of a single 5 s RNA molecule (Knight & Darnell, 1967).

(4) The 45 s RNA is generally considered to be a precursor of both 28 and 18 s ribosomal RNA. Since the molecular weights of the latter two RNA’s total only 2.6 x 10’ daltons, it follows that the 45 s molecule includes some RNA that is not a precursor of 18 or 28 s RNA.

Of course, the 45 s RNA must also be a precursor of the 32 s RNA, if the latter be accepted as a precursor of 28 s RNA. Even so, the sum of the molecular weights of the 32 s and 18sRNA’s (3.1 x 106) is much less than the molecular weight of the 45 s RNA.

We therefore conclude that one third to one half of the 45 s molecule is not incor- porated into the 18 or 28 s ribosomal RNA.

(5) On the basis of their studies of the methylation of ribosomal RNA precursors in HeLa cells, Weinberg et al. (1967) h ave also concluded that the processing of ribosomal RNA precursors is non-conservative. Their data suggest that the non- conserved portions of the 45 s molecule are non-methylated. Cur own unpublished data on the steady-state levels of methylation of these molecules agree with Weinberg et al. (1967). Since the absence of methylation may be a general property of messenger RNA (Moore, 1966), we are led to wonder whether the non-conserved portion of 45 s RNA might serve as messengers for some of the structural proteins of the ribosome, or for enzymes involved in ribosome biosynthesis.

This work was done in 1966 while the authors were associated with the Biological Laboratories at Harvard University. It was supported by a National Institutes of Health

550 E. H. McCONKEY AND J. W. HOPKINS

grant no. GM07606 to one of us (J.W.H.) and by a National Science Foundation grant no. 1254 to Professor J. D. Watson. We thank Professor A. M. Pappenheimer, Jr. for the use of research space, Professor Paul Doty for the use of the analytical ultracentrifuge, and Mr Malcolm Smart for his generous assistance with the centrifugation.

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