Small molecular weight monodisperse nuclear RNA

J. Mol. Biol. (1968) 38, 289-304

Small Molecular

Weight Monodisperse

Nuclear RNA

ROBERT A. WEINBERG AND SHELDON PENMAN

Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 01239, U.X.A. (Received 26 August 1968) The nuceloplasm and nucleolus of HeLa cells contain six distinct low molecular weight species of RNA. Their size, as estimated from electrophoretic mobility, ranges from 100 to 180 nucleotides. These RNA species are long-lived and do not appear to be precursor to any cytoplasmic product. At least four of them are extensively methylated. Their base compositions range from 47 to 54% guanosine + cytidine. Similar species of nuclear RNA are found in mouse fibroblast cultures and in the developing chick embryo brain.

1. Introduction in vertebrate cells have indicated that in addition to nucleolar ribosomal precursor RNA (Edstrom, 1961; Perry, 1962; Chipchase & Birnstiel, 1963; Muramatsu, Hodnett & Busch, 1966; Weinberg, Loening, Willems & Penman, 1967), the nucleus contains a class of large molecular weight, heterodisperse RNA of unknown function (Scherrer, Marcaud, Zajdela, London & Gros, 1966; Attardi, Parnas, Hwang & Attardi, 1966; Houssais & Attardi, 1966; Warner, Soeiro, Birnboim, Girard & Darnell, 1966; Penman, Smith & Holtzman, 1966). In this report, we will describe still other types of RNA, associated with the nucleoplasm and nucleolus. The existence of this new class of RNA was first suggested by the work of Knight & Darnell (1967), in which acrylamide gel electrophoresis of nuclear RNA unexpectedly revealed the existence of previously uncharacterized low molecular weight species. While the present work was in progress, two additional reports appeared confirming the existence of such low molecular weight nuclear RNA (Dingman & Peacock, 1968; Nakamura, Prestayko & Busch, 1968). The present report presents detailed characterization of these species and describes their localization within the nucleus. The nuclear RNA species described here are different from any other previously characterized class of nuclear RNA. The nuceloplasm has been shown to contain heterodisperse RNA, distributed in size from 10 to 90 s. The nucleolar fraction contains precursors to ribosomal RNA which range in size from 18 to 45 s. When the high molecular weight RNA’s of the nucleolus and nucleoplasm are labeled, the radioactivity is only transiently associated with the nucleus, since the nucleolar RNA is precursor to cytoplasmic ribosomes and the nucleoplasmic heterodisperse RNA is metabolically unstable. In contrast, the nuclear RNA species described here range in size from 4 to 6 S, and appear to be stably associated with the nucleus. Some are associated principally with the nucleoplasmic and others with the nucleolar Recent

investigations

of RNA metabolism

289

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R. A. WEINBERG

AND

S. PENMAN

fraction. These species are methylated and have base compositions unlike any of the other nuclear RNA classes.

2. Materials and Methods (a) Cells Type 3 HeLa cells were grown in suspension culture harvested at a concentration of about 4 x lo5 cells/ml.

as previously described. (Eagle, 1959).

Cells were

(b) Radioisotopes For periods of incorporation of 2 hr or less, cells were concentrated to 2 x lo6 cells/ml. For long periods of incorporation of 16 hr or more, cells were diluted to 2 x lo6 cells/ml. The methyl groups of RNA were labeled by the addition of Lj3H-methyllmethionine to the medium. The medium contained guanosine and adenosine at a concentration of lo-*M to suppress the incorporation of radioactivity from methylmethionine via onecarbon transfer into pukes. Phosphate-free Eagle’s medium was used for pulse-labeling cells with 32P. Cells were washed several times in this medium before addition of carrierfree Na332P04. Following 30 min of exposure to 32P04, cells were resuspended in unlabeled medium and chased for 48 hr. For long continuous 32P04 labeling, Na332P04 was added to regular culture medium. Sterile carrier-free Na332P04 was purchased from Isoserve, Cambridge, Mass. [2J*C]Uridine (5Omc/m-mole) and [5-3H]uridine (20 mc/ pmole) were purchased from Schwarz Biochemical. L-[3H-methyZ]Methionine (2 mc/pmole) was purchased from the Nuclear Chicago Corp. (c) CeZZfractionation The fractionation of HeLa oells into cytoplasm, nucleoplasm and nucleolar fractions haa been previously described (Penman et al., 1966). Briefly, the cells are washed and resmpended in hypotonic buffer (RSB: 0.01 m-NaCl, 0.003 M-MgCl,, 0.01 i@-Tris, pH 7.4), and allowed to swell for 5 min. The cells are then ruptured in a precision-bore, stainless steel Dounce homogenizer, and the crude nuclei are deposited by centrifugation. The nuclei are washed once more in RSB buffer and then resuspended in RSB buffer and treated with a detergent mixture of Tween 40 and sodium deoxycholate. The nuclei, which now have their outer nuclear membrane removed, are deposited again by centrifugation and the supernatant pooled with two previous supernatants. These 3 pooled supernatants are termed the cytoplasmic fraction. When desired, ribosomes are prepared from the first cytoplasmic supernatant by the addition of sodium deoxycholate to 0.5% and dextran sulfate to 1 mg/ml. The ribosomes are pelleted by centrifugation for 90 min at 40,000 rev./min in the Spinco no. 40 rotor. The nuclear pellet is digested by resuspending the purified nuclei in high salt buffer (HSB: 0.5 m-NaCl, 0.05 M-MgCl,, 0.01 M-Tris, pH 7.4), adding electrophoretically pursed DNase, and incubating at 37’C for a few minutes. After the complete dispersion of clumped chromatin, the digested nuclei are then separated into nucleolar and nucleoplasmic fractions. The best separation of nucleoli and nucleoplasm is accomplished by zonal centrifugation (Willems, Wagner, Laing & Penman, 1968). The nuclear digest is layered on a 15 to 30% sucrose gradient made with HSB buffer and centrifuged for 15 min at 22,000 rev./min in the 25.3 rotor of the Beckman-Spinco ultracentrifuge. (d) Preparation

of RNA

The pellet obtained after zonal centrifugation consists principally of nucleoli and is resuspended in sodium dodecyl sulfate buffer (0.1 M-N&~, 0.01 M-Tris, 0.001 M-EDTA, 0.5% sodium dodecyl sulfate, pH 7.4) before phenol extraction. The nucleoplasmic contents are spread throughout the sucrose gradient, which is decanted from the centrifuge tube and precipitated with 2 vol. of ethanol. The nucleoplasmic ethanol precipitate is centrifuged and the pellet is resuspended in sodium dodecyl sulfate buffer and extracted with phenol as above. The cytoplaamio fraction is also extracted with phenol after

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addition of EDTA to O-01 M and sodium dodecyl sulfate to 0.05%. The hot phenolsodium dodecyl sulfate extraction procedure was first described by Scherrer 62 Darnell (1962) and modified by the addition of chloroform (Penman, 1966), and the reduction of temperature to 55°C (Wagner, Katz & Penman, 1967). Following phenol extraction, samples are precipitated with 2 vol. of ethanol. The DNA present in the nucleoplasmic fraction interferes with subsequent analysis by gel clectrophoresis. Therefore, following ethanol precipitation, the pellet is resuspended in 0.2 ml. of 10 x RSB and incubated at 37°C with 20 pg of electrophoretically purified DNase for 1 min. The solution is reprecipitated with ethanol and centrifuged. When RNA is to be analyzed by gel electrophoresis, the ethanol precipitates are dried under vacuum and resuspended in 50 ~1. of electrophoresis buffer (0.04 iw-Tris, 0.02 M-sodium acetate, 0.002 M-EDTA, 0.2% sodium dodecyl sulfate, pH 7.4). (e) Acrylamide

gel electrophoreeie

The technique used for acrylamide gel electrophoresis of RNA has been described (Loening, 1967; Weinberg et al., 1967). Gels are cast with 10% glycerol to facilitate freezing and slicing. The same technique used previously for gels of 2.6% acrylamide content (Weinberg et al., 1967) is used here for gels containing 10% and 15% acrylamide. The concentration of cross-linking agent (ethylene diacrylate) is 0.25% (v/v) in all types of gels. Electrophoresis is performed at 10 v/cm for the time specified. Following electrophoresis, the gels are scanned for optical density, frozen, and sliced into l-mm sections. The sections are hydrolyzed for several hours with 0.4 ml. of concentrated NH,OH in closed scintillation vials. The water-miscible scintillant used here is composed of 6 parts toluene scintillator and 4 parts methoxyethanol. ( f) Base composition analysis Base composition analysis was performed as described in detail in Willems et al., 1968, and Wagner & Ingram, 1966.

3. Results (a) Small molecular weight RNA content of the different cell fractions The optical density patterns of acrylamide gels following electrophoresis of the large and small molecular weight RNA species of the various cell fractions are shown in Figure 1. The ordinates on each profile suggest the relative amounts of each RNA species in HeLa cells. The large molecular weight RNA was analyzed on 2.6% gels and the optical density patterns are presented in the left column. In the right column, 10% gels of small molecular weight RNA species are shown. The subject of this report is the RNA species seen in the optical density profiles of small molecular weight RNA of the nucleoplasmic and nucleolar fractions as shown in Figure l(e) and (g). These nuclear species (Fig. l(e) and (g)) are composed of RNA in that they are labeled by 32P0, and [3H]uridine, are resistant to degradation by pronase and DNase, and are degraded by RNase and alkali. The products produced after alkali degradation behave as monoribonucleotides upon high-voltage paper electrophoresis (R. A. Weinberg, unpublished observations). The number of molecules of the most prominent of the nucleoplasmic species shown in Figure l(e) is equal to one-fifth the number of ribosomal 5 s RNA molecules present in the cytoplasm. Figure 1 also demonstrates that the nucleoplasm and nucleolus together contain a large pool of ribosomal5 s RNA, in accord with the results of Knight & Darnell(1967). The cytoplasmic small molecular weight non-ribosomal RNA (Fig. l(b)) seems to consist solely of transfer RNA (4 s), but the existence of small amounts of other species cannot be excluded. Careful examination of the cytoplasmic fraction has failed to reveal the existence of small molecular weight RNA species resembling those characterized here in the nuclear portion of the cell.

292

R. A. WEINBERG

AND

2.6 % Acrylamide

S. PENMAN 10% Acrylamide

(a)

1

2

3

4

Distance

2 migrated

3

4

(cm)

FIG. 1. RNA was prepared from cytoplasmic, nucleoplasmic, and nucleolar fractions of lOa cells. One-half of each preparation was analyzed for high molecular weight species (2.6% acrylamide gels) and one-half for low molecular weight species (10% aorylamide gels). Following eleotrophoresis, the optical density profile of each gel was measured using a Gilford recording speotrophotometer adapted for this purpose (Weinberg et al., 1967). All gels shown here were run for approximately 4 hr. Ordinates are expressed in an arbitrary unit proportional to optical density. (a) High mol. wt. RNA from whole cytoplasm; (b) low mol. wt RNA from purified ribosomes; (c) low mol. w-t non-ribosomal oytoplasmio RNA; (d) high mol. wt nuoleoplasmio RNA; (e) low mol. w-t nuoleoplasmio RNA; (f) high mol. wt nuoleolar RNA; (g) low mol. wt nuoleolar RNA.

The small molecular weight ribosomal fraction (Pig. l(c)) contains a small amount of transfer RNA (labeled 4 s), the 5 s ribosomal RNA, and in addition, the 28 sA RNA, discovered recently by PQne, Knight & Darnell (1968). They have shown that this species is normally hydrogen-bonded to the 28 s ribosomal RNA and remains associated with the 28 s RNA during cold phenol extraction. It is melted free of the 28 s RNA upon heating to temperatures used in hot phenol extraction. This RNA exhibits both the aedimentation velocity and electrophoretic mobility of a species of approximately 5-5 S. The term 7 s originally given to this species by P&e et al. (1968) is thus a misnomer, and for want of a more appropriate name, we shall term this species the 28 s associated or simply 28 sA. This preparation (Pig. l(c)), derived from ribosomes and consisting of 4 S, 5 s and 28 sA RNA’s, will be used as reference marker in subsequent gels. (b) The two forms of ribosomal 5 s RNA Detailed inspection of the 5 s peak (Fig. l(c)) of the RNA derived from ribosomes reveals a minor, more rapidly migrating shoulder. This shoulder has been observed

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RNA

293

by others (Aubert, Monier, Reynier & Scott, 1967; Hindley, 1967; Forget & Weissman, 1967), and represents a reversibly denatured form of 5 s ribosomal RNA generated by hot phenol extraction. In Figure 2, tritium-labeled 5 s RNA which was phenolextracted at room temperature has been co-electrophoresed with 14C-labeled hot phenol (55°C)extracted 5 s ribosomal RNA. The physical chemistry of this reversible denat’uration will be reported by others (R. Monier, personal communication). We

Slice no.

FIG. 2. Ribosomes were isolated from 5 x 10’ cells labeled 24 hr with 100 pc of [eH]uridine and from 5 x lo7 cells labeled 24 hr with 2 PC of [‘*C]uridiue. Ribosomal pellets were dissolved in 1 ml. of sodium dodecyl sulfate buffer and centrifuged 16 hr at 25,000 rev./min at 25’C in the SW25.3 rotor of the Spiuco L2-50 ultracentrifuge. Material sedimenting as far a8 8 s was pooled in each gradient. 3H material was extracted with phenol at 25°C. I%‘-labeled material was extracted with phenol at 55’C. The preparations were then pooled and electrophoresis was run on a 10% acrylamide gel for 9 hr. The 5 s region of the gel is shown here. -*--a-, [rV]Uridine; --O-O-, [3H]uridine.

mention this conversion here, since in subsequent gel profiles, the 5 s peak will frequently be split into two components in varying proportions. This b&modal profile should therefore not be interpreted either as error in slicing the gels, or as representative of two physiologically differing states of 5 s ribosomal RNA. It is only an artifact of hot phenol extraction, and the varying proportions of the two conformations reflect solely the effects of heating and magnesium concentration during preparation. (c) Electrophoretic rnobilities of the nuclear RNA species The total nuclear RNA (nucleoplasmic and nucleolar) from cells labeled with [3H]uridine and harvested after 24 hours growth was co-electrophoresed with 14Clabeled (ribosome-derived) marker. The result is shown in Figure 3. The peaks in the nuclear material have been labeled A to I. Peaks G and G’ migrate with the (ribosomederived) 5 s RNA. The broad peak labeled I corresponds to transfer or 4 s RNA, and is barely detectable. A peak corresponding to 28 s associated RNA (28 sA) is always

294

R. A. WEINBERG I

I

I

AND I

S. PENMAN I

I

I

Slice no.

FIG. 3. Whole nuclear RNA (nuoleoplasmic + nucleolaz) was prepared from 5 x IO7 cells which had been labeled 24 hr with 100 PC of r3H]uridine. Ribosomes were prepared from an equal number of cells grown 24 hr in the presence of [14C]uridine. 14C-labeled ribosomal marker was mixed with the 3H-labeled nuclear RNA, the sample was layered onto a gel of 1.5% acrylamide, and electrophoresis was performed for 18 hr. -O---O--, sH; -.--a---, 14C.

in the nucleus but in very low amounts. The two small molecular weight ribosomal structural RNA species (peaks E and G + G’) are present in equimolar amounts in the cytoplasmic ribosomes. In the nucleus, 5 s (peaks G + G’) is present in great excess relative to 28 SA (peak E), which is consistent with the existence of a sizeable pool of 5 s ribosomal RNA (peaks G + G’) in the nucleus. The two conformations of 5 s RNA are present in differing proportions in the marker (“C) and in the nuclear preparation (3H), a reflection of the fact that these two preparations were phenolextracted separately. The reproducibility of the distribution of nuclear RNA species justifies the adoption of a preliminary nomenclature (species A-I). All are well defined with the exception of species I which is consistently present as a broad band, not obviously identical to cytoplasmic transfer RNA. Of the remaining species, two (E and G + G’) are known to be present in large amounts in the cytoplasm. Their presence in this preparation probably reflects their transient existence in the nucleus en route to the cytoplasm, although such a precursor-product relationship is not proved here. There are six homogeneous species (A, B, CD, F and H) which are reproducibly present in the nuclear preparation and which are not found in detectable amounts in the cytoplasm. It should be noted that only relatively long-lived species of RNA are labeled in this preparation as a consequence of the 24 hours between the addition of label and the harvesting of the cells. In addition, the use of uridine of high specific activity results in an early depletion of the labeled compound in the medium, causing further suppression of label in short-lived species in this preparation.

MONODISPERSE

NUCLEAR

RNA

295

(d) Partition of species between nudeoplasm and nucleolus The partition of the low molecular weight RNA between the nucleoplasmic and nucleolar fractions is shown in Figure 4. Nucleolar RNA labeled with 3H and nucleoplasmic RNA labeled with l*C were co-electrophoresed. It is apparent that the major species A and the minor species B and F are found in the nucleolar fraction. The remaining nuclear species can be found in both nuclear fractions. Species E (28 sA) and G + G’ (5 S) constitute a special class among this low molecular weight RNA since they are precursors of components of cytoplasmic ribosomes and are transiently associated with the nuclear fractions. A meaningful comparison of the relative amounts in the two fractions is not possible unless identical labeling conditions are used. Figure 4 does not present accurately the relative molar amounts of the various RNA species in the nucleoplasmic and nucleolar fractions since different labels were used. A more accurate measure of the relative molar amounts of these nuclear species is presented in Table 1. The amounts listed there represent the estimated steadystate number of molecules per cell as computed from optical density tracings of acrylamide gels and estimated molecular weight of each species. These molecular weight estimates, also shown in Table 1, were made assuming a logarithmic relationship between migration rates and molecular weights (Bishop, Claybrook & Spiegelman.

200 -

ISO4

2 5 :: 2 Y IOOb 0I

50-

Slice no.

FIG. 4. Nucleolar RNA was prepared aa described in Materials and Methods from 5 x 107 cells grown 24 hr in the presence of 100 pc of [3H]uridine. Nucleoplasmio RNA was prepared from 5 x IO’ cells grown 24 hr in the presence of 2 PC of [l*C]uridine. The RNA preparations were extracted separately, pooled, and run 9 hr on a loo/, acrylamide gel. -O--O-, 3H; -O-O-, 14C.

296

R. A. WEINBERG

AND TABLE

Species of RNA

A B C (28 sA) (5 9) (tRNA)

: G:nd ;) 18 s 28 s 32 s 45 s

G’

Estimated

Cytoplasm 5x106 5x100 1 x 108 5x106 5x106 -

S. PENMAN

1

Estimated no. of nucleotides

no. of molecules/cell

Nuoleoplasm 4x105 1x106 5x104 3x105 2x106 2 x 106’

Nucleolus 2 x 105 1 x 104 1x106 7 x 104 1 x 104 3x104 3x105 1 x 105 6 x 1040 7 x 1040

-

4x104 1x104

180 170 165 150 140 125 1218 100 80” 1.8 x 10s6 4.9 x 1036 6.8 x 103e 1.3 x 1048

*Forget & Weissman, 1968. bDayhoff t Eck, 1968. CThe in wivo partitioning of 18 and 28 s ribosomal RNA between nucleolus and nuoleoplrtam is not clear. because of the DNaae-high salt method used to release the nucleoli. aPeterislnn I% Pavlovec, 1966. OWeinberg et al., 1967. ‘Species I includes a diffuse band of material which migrates with the approximate electrophoretic mobility of tRNA. #Estimates obtained from O.D. tracings of the type shown in Fig. 1 and are accurate to within 20%.

1967). The relative migration rates are quite reproducible from one gel to another. The estimation of molecular size may be slightly inaccurate due to special conformational states which some of these species may assume. More accurate determinations must await precise biochemical analysis. Where noted, molecular weight estimations are from already published material. On a molar basis, species A, B and P appear to be exclusively nucleolar. Species C and D are found principally in the nucleoplasm. Species H occurs in comparable amounts in both nuclear fractions. The amounts of various other cellular RNA species are included to suggest the relative number of molecules of all of these species in the HeLa cell. The partition of the species of low molecular weight RNA between the nucleoplasmic and nucelolar fractions may reflect true physiological compartmentalization, or alternatively, may result in part from the techniques used here in nuclear fractionation. These techniques have been proved for the high molecular weight nuclear RNA species (Willems et al., 1968), but are still to be justified for the low molecular weight RNA. The nuclear fractionation was also carried out under conditions of higher ionic strength (0.8 M- instead of O-5 M-NaCl). This modified technique produces a nucleolar preparation almost completely free of high molecular weight nucleoplasmic contamination (Penman, Vesco & Penman, 1968), but the resultant partition of small molecular RNA is identical to that in Figure 4. We have not investigated alternative techniques to separate the nucleoplasm from the nucleolus.

MONODISPERSE

NUCLEAR

297

RNA

(e) Metabolic stubility During short labeling periods, the 4 to 6 s regions of both the nucleolar and nucleoplasmic preparations contain a large amount of heterodisperse RNA which is metabolically unstable in contrast to the monodisperse species discussed here. Because of this heterodisperse material, it is diEicult to measure the kinetics of labeling of the monodisperse species for short periods. It is possible, however, to compare the general features of the pattern of radioactivity obtained after one hour label with those of material which has been labeled and then chased for 48 hours. Figures 5 and 6 show the results of such experiments. Cells were exposed to medium containing Na,32P0,, removed from this medium, and grown for 48 hours in normal, non-radioactive medium. The cells were pulsed for one hour with [3H]uridine immediately before harvesting. Both the nucleoplasmic (Fig. 5) and nucleolar (Fig. 6) fractions were analyzed using 2.6% gels to display the pattern of high molecular weight RNA, and 10% gels to display that of low molecular weight RNA. The high molecular weight, heterogeneous, nucleoplasmie RNA (Fig. 5, lower left) is highly labeled during the tritium pulse. The RNA labeled with 32P and chased shows only some ribosomal RNA and a small amount of low molecular weight material. This ribosomal RNA (18 and 28 s) represents a very small fraction of the 600 D

2 .

2000-

E 2

;

isoo-

0

IOOO-

500

-

20

40

60

80 Slice

100

120

no.

FICA 5. 5 x lo7 cells were grown in a normal growth medium in the presence of 2 mc of Na,32P0, for 24 hr. At that time, they were transferred to fresh medium without label and &lowed to grow for 48 more hours, at which tune they were concentrated 10 times and exposed to 200 PC of [aH]uridine for 1 hr. The cells were then harvested and the nucleoplasmic RNA extracted. 20% of the nucleoplasmic RNA was run on a 2.6% gel for 5 hr (lower left). The remaining 800/& was run on a 10% gel for 9 hr (insert at upper right). The brackets indicate the region of the 2.6% gel which was analyzed in detail on the 10% gel. The ordinates have been adjusted to compensate for the amounts of material layered onto each gel. -O-O-, 3H; -a-a---, eaP.

298

R. A. WEINBERG

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S. PENMAN

-2 .1000 5 500

Slice

,v a ::

no.

FIG. 6. 5 x 10’ cells were washed 3 times in a medium lacking phosphate, and then incubated for 30 min in 10 ml. of medium lacking non-radioactive phosphate but containing 2 mc of carrierfree NacszPO The cells wzre then pelleted and resuspended in normal growth medium. After 48 hr of further growth, the cells were concentrated 10 times and labeled with 100 PC of [aH]uridine for 1 hr. The cells were then harvested and the nucleolar fraction prepared. 10% of the nucleolar RNA was layered onto a 2.6% gel and allowed to migrate for 3.5 hr (lower left). 90% of the nucleolar RNA was layered onto a 10% gel which was run for 9 hr (insert at upper right). The brackets indicate the region of the 2.6% gel which is analyzed in detail on the 10% gel. The ordinates have been adjusted to compensate for the amounts of material layered onto each gel. --O-O-, 3H; -.--.-, sap.

total cellular ribosomal RNA and may represent cytoplasmic contamination. The low molecular weight portion of the 2.6% gel is examined in detail on the 10% gels (Fig. 5 inset, upper right). This low molecular weight RNA region contains a large amount of unstable tritiated material which is not labeled after a long chase (32P). Several of the peaks labeled during the tritium pulse seem to coincide with the more stable 32P-labeled material. The 2.6% acrylamide gel profile of 3H pulse-labeled high molecular weight nucleolar RNA (Fig. 6, lower left) shows the 45, 32 and 28 s ribosomal precursors and very little low molecular weight material. Following a 48-hour chase, the high molecular weight material has disappeared completely, and there is a barely discernible amount of stable material in the 4 to 6 s region. The low molecular weight material is again resolved on a 10% gel (Fig. 6, inset, at upper right). During a short pulse of 3H, only two of the low molecular weight species are clearly labeled in the nucleolus. The most prominent is the 5 s ribosomal, separated here into its two conformers (species G + G’). Also, species D is highly labeled. The longer labeled, chased material (““P) shows the same profile as that seen for the nucleolar fraction in Figure 4.

MONODISPERSE

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RNA

(f) Base composition The base composition of the prominent small molecular weight nuclear species has been measured. Cells were pulse-labeled with 32P and chased for 48 hours as described in Materials and Methods. The radioactivity profiles obtained exhibited a significant heterogeneous background, whose base composition is fairly constant throughout the gel. The base compositions presented in Table 2 have been corrected for contaminating underlying material. The base compositions of the different species vary significantly. This is consistent with each species being a unique type of RNA. TABLE

2

Species

o/oAdo

% CYd

o/oGuo

o/oUr d

% Guo + Cd

A c D E (28 sA)

22.5 21.4 18.4 17.8 21.5 18.2 18.2 26.0 19.1

19.6 20.4 25.0 27.4 26.3 27.3 27.3 17.3 25.5

28.5 27.1 29-l 30.7 29.8 30.0 32.2 30.0 33.7

29.3 31.2 27.4 24.1 22.4 24.0 22.3 26.7 21.7

48.1 47.5 54.1 58.1 56.1t 57.3 59q 47.3 59.2

G(6 s) H I

t PBne, Knight C%Darnell, 1968. $ Forget & Weissman, 1968.

(g) Methyhtion The small molecular weight nucleoplasmic and nucleolar species are also methylated to different extents. The extent of methylation was measured by exposing cells to [3H-methyZ]methionine and 32P0, simultaneously for periods of up to a day. The ratio of [3H]methyl to 32P0, counts represents a more accurate measurement of the amount of methylation than does [3H-methyl]methionine to [l*C]uridine. The latter technique depends upon knowledge of the base composition of a given species. Also, labeling with 32P04 and [3H-methyZ]methionine in a medium containing cold phosphate and cold methionine assures continuous linear uptake of label over the entire labeling period. Measurement of the extent of methylation implies measurement of the number of methyl groups which have been joined to the RNA molecule subsequent to transcription on the DNA template. Care must therefore be taken to prevent radioactivity from labeled methyl groups from entering other moieties besides methyl groups attached to the ribose or bases. In particular, the entry of radioactivity from methyl groups into the purine skeleton via the one-carbon transfer will introduce an inaccuracy into measurement of the amount of methylation. In these experiments, the entrance of label into the purine biosynthetic pathway was suppressed by addition of adenosine and guanosine to the medium. In other experiments, not reported here, purine biosynthesis was inhibited by the use of aminopterin supplemented with hypoxanthine, thymidine and glycine. The amount of non-specific label

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R. A. WEINBERG

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S. PENMAN

entering RNA was measured by ascertaining the amount of tritium associated with ribosoma15 s RNA. Since ribosoma15 s RNA is not methylated (Forget & Weissman, 1968), any tritiumradioactivity associatedwith this species is due tonon-specificlabeling. In all of the methylation experiments, some 3H radioactivity was found associated with the unmethylated 5 s RNA. In the experiment whose results are displayed here in Figure 7, the [3H]methy1/32P0, ratio of the 5 s ribosomal RNA was sufficiently low to allow for accurate determination of the extent of methylation of the major small molecular weight nuclear RNA species. After correcting for “non-specific” tritium labeling, the number of methyl groups in each species was estimated and the results are presented in Table 3. The nuclear RNA species measured all appear to be extensively methylated. TABLE 3

source

3H/32P

3H/3aP ratios x estimated no. of nucleotides

A C

Nucleolus Nucleolus

0.135 0.257

15.0 33.8

D

Nucleoplasm Nucleolus

0.270 0.189

36.0 20.6

G(5 s) H

Nucleoplasm Cytoplasm Cytoplasm Nuoleolus

0.181 0.052 0.040 0.295

19.4 -

4s

Nucleoplasm Cytoplasm

0.287 0.508

23.5 36.6

Species

Estimated no. of methyl groups/ molecule 3 6

3 E (28 sA)

-

24.3 4

t Personal communication (L. Culp & $ 0.05 was subtracted from all numbers counts. The corrected 3H/3aP ratio for length in nucleotides of that species to groups associated with the species.

6.5.f

G. M. Brown). in the third column to correct for “non-specific” tritium a given species was then multiplied by the estimated give a product proportional to the number of methyl

Examination of the nucleoplasmic profile (Fig. 7(a)) reveals an intensely methylated component migrating in the region of species G’. The nucleolar profile (Fig. 7(b)) demonstrates a methylated component migrating slightly behind species G‘. A comparison of these two peaks with that of the ribosomal RNA (Fig. 7(c)) from the same cells suggests the existence of yet another species of nuclear RNA which is intensely methylated, present in very small amounts, and not separable from the 5 s (species G + G’) RNA by the acrylamide gel technique used here. This intensely methylated species is undetectable by other labeling techniques and is associated principally with the nucleoplasm. (h) Inter-species comparison Figure 8 shows the results of co-electrophoresis of whole nuclear RNA from chick embryo brain with whole nuclear RNA from HeLa cells. The profiles are quite similar, although one shoulder appearing in the chick profile has no counterpart in HeLa cells. The significance of the profiles of Figure 8 will be discussed below.

MONODISPERSE I

600 - (a)

NUCLEAR /

,

I (b)

Nucleoplasmic

I

301

RNA I c

Nucleolar

x70

90

II0

130 Cc)

Slice no. 84 ’

Ribosomal

I ! i800

nil

2 2

Slice no. FIG. 7. 4 x lo7 cells in 100 ml. of norm81 medium were grown up to 8 x 10’ cells in the presence of 1.2 mc of L-[3H-methyZ]methionine, 4 mc of Na,3aP0,, 10d4 an-adenosine and IO-* M-gua~~osine. The RNA shown in e8ch part of this Figure was run 9 hr on a 10% acrylamide gel. (a) Nucleoplesmic; (b) nucleoler; (c) ribosomal. -O-O--, 3H; -@-a-, 3aP.

4. Discussion This report describes several species of monodisperse RNA of low molecular weight confined to the nucleus of HeLa cells. These species represent a very small fraction of total cellular RNA (about O-4o/o). Table 1 shows, however, that the number of molecules is considerable. There are about one-fifth as many molecules of species D as there are ribosomes in the cell. These species appear to be relatively stable. They are still labeled after a 4%hour chase (Figs 5 and 6) and clearly are longer-lived than any other nuclear RNA. Experiments comparing labeling before and after a chase indicate lifetimes of at least several days in cells whose generation time is 24 hours (R. A. Weinberg, unpublished results). Since the lifetime of the low molecular weight nuclear RNA appears longer than one generation time, these species must survive mitosis and re-associate themselves with the nucleus upon re-formation of the nuclear envelope. Species A,B, C,D,F and H seem to be unrelated to cytoplasmic transfer and ribosomal RNA. Species E and G+G’ are precursors to cytoplasmic small molecular weight ribosomal RNA. The relation of species I to cytoplrtsmic RNA is unclear. Species I is characterized by a broad distribution similar to that of the cytoplasmic 21

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Slice no

FIG. 8. Four 14-day old chicken embryos, each of which had been inoculated on the 10th day with 100 PC of Na,3aPOI, were kindly provided by Dr David K&bat. The whole brains were excised, washed briefly in Earle’s saline and then placed in 5 ml. of a modified RSB buffer contaming 0.14 rd-NaCl instead of 0.01 M-N&I. The whole brains were homogenized in this medium with a “B” Dounce homogenizer made by the Kontes Glass Co., Vineland, N.J. The nuclei were pelleted and resuspended and pelleted once more. The nuclear pellet was then suspended for 1 mm in 1 ml. of normal RSB buffer to lyse erythrocytes. 6 ml. of the modified (0.14 M-N&I) RSB was then added and the suspension of nuclei w&s made 1.0% in the detergent Nonidet P40. The nuclei were pelleted and resuspended in HSB buffer. Following DNase digestion, the nuclear fraction was precipitated with ethanol, resuspended in 2 ml. of sodium dodecyl sulfate buffer and extracted with phenol. The 3aP-labeled whole nuclear RNA from chickens W&B mixed with [3H]uridine-labeled whole nuclear RNA from HeLa cells which had been grown 24 hr in the presence of radioactivity. Electrophoresis was performed on a 10% gel for 9 hr. --O-O-, 3H; -.-a--, =P.

transfer RNA. It may in fact result from a small contamination of the nucleoplasm by the cytoplasmic fraction. Species I is in any case not the precursor to cytoplasmic tRNA. The precursor to cytoplasmic tRNA has recently been reported (Burdon, Martin & Lal, 1967). Preliminary observations in this laboratory (R. D. Leibowitz & R. A. Weinberg, unpublished observations) and a more detailed report (D. Bernhard & J. E. Darnell, manuscript in preparation) indicate that the precursor to cytoplasmic tRNA appears in the cytoplasm within minutes after synthesis. The electrophoretic mobility of this tRNA precursor is different from any of the species shown in Figure 3. Past investigations of nuclear preparations similar to those presented here suggested that all ribosomal RNA found in the nucleus is either in transit to the cytoplasm or present as oytoplasmic contamination, i.e. that there are no functioning ribosomes in the HeLa cell nucleus (Penman, 1966). Thus the small molecular weight nuclear RNA does not appear to be involved in normal protein synthesis requiring ribosomes. Most, if not all, of these nuclear RNA species are methylated, confirming a similar finding of Knight & Darnell (1967). Besides ribosomal and transfer RNA, a third class of cellular RNA must now be considered as substrate for methylating enzymes. The finding of labeled 5 s ribosomal RNA (species G and G’) in the nucleolus following a 4%hour chase (Fig. 7) is consistent with the 6nding previously reported (Knight & Darnell, 1967) that a large pool of 5 s ribosomal RNA exists in the nuclear

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fraction. In the absence of such a pool, 5 s ribosomal RNA would behave kinetically like 28 s associated RNA (species E), since both are present in equimolar amounts in cytoplasmic ribosomes. This study has not attempted to determine the nature of the association between these small molecular weight nuclear RNA species and the nucleoprotein complex known to exist in the nucleus. The fact that these species are unaffected by pronase or DNase would indicate that they are probably not covalently linked to protein or DNA in wivo. In addition to the chicken-HeLa comparison demonstrated in Figure 8, recent work in this laboratory indicates similar nuclear RNA in L-cells (R. A. Weinberg, unpublished observations) and in the 3T3 mouse fibroblast line (A. Rein & R. A. Weinberg, unpublished observations). In all these cases,the relative proportions of the various species resemble those of HeLa cells, but the electrophoretic mobilities of the various species differ in some cases from their counterparts in HeLa cells. These results, together with the previously cited reports (Dingman & Peacock, 1968; Nakamura et al., 1968), imply that these species of RNA are not confmed to HeLa cells, and probably represent a general phenomenon of vertebrate and perhaps rucaryotic cells. This work was supported by awards CA08416-02 from the National Institutes of Health and GB4320 of the National Science Foundation. One of us (S.P.) is a career development awardee of the U.S. Public Health Service, GM16127-02. The other author (R.A.W.) is a pre-doctoral fellow of the National Institutes of Health, grant Fl-GM-23, 89803. The capable assistance of Irene Fournier and Maria Penman is gratefully acknowledged. Note added in proof: Further experiments indicate that species G’ consists of a mixture of the denatured conformer of 5 s ribosomal RNA and a co-migrating stable nucleoplasmic RNA. REFERENCES Attardi, G., Parnas, H., Hwang, M. H. & Attardi, B. (1966). J. Mol. Biol. 20, 145. Aubert, M., Monier, R., Reynier, M. & Scott, J. F. (1967). In Proc. 4th FEBS Meeting, vol. 3, p. 172. Oslo: Universitets Forlaget. Bishop, D. H. L., Claybrook, J. R. & Spiegelman, S. (1967). J. Mol. Biol. 26, 373. Burdon, R. H., Martin, B. T. & Lal, B. M. (1967). J. Mol. Bill. 28, 357. Chipchase, M. I. H. & Birnstiel, M. L. (1963). Proc. Nat. Acad. Sci., Wash. 49, 692, Dayhoff, M. 0. & Eck, R. V. (1968). Atlas of Protein Sequence and Structure. Silver Spring, Maryland: National Biomedical Research Foundation. Dingman, C. W. & Peacock, A. C. (1968). Biochemistry, 7, 659. Eagle, H. (1959). Science, 130, 432. Edstrom, J. E. (1961). J. Biophy8. Biochem. CytoZ. 11, 549. Forget, B. G. & Weissman, S. M. (1967). Nature, 213, 878. Forget, B. G. & Weissman, S. M. (1968). Science, 158, 1696. Hindley, J. (1967). J. Mol. BioZ. 30, 126. Houssais, J. F. t Attardi, G. (1966). Proc. Nat. Acad. Sk., Wash. 56, 616. Knight, E., Jr. & Darnell, J. E. (1967). J. Mol. BioZ. 28, 491. Loening, U. (1967). Biochem. J. 102, 251. Muramatsu, M., Hodnett, J. & Busch, H. (1966). J. BioZ. Chem. 241, 1544. Nakamura, T. Prestayko, A. W. & Busch, H. (1968). J. BioZ. Chem. 243, 1368. Pene, J. J., Knight, E., Jr. & Darnell, J. E., Jr. (1968). J. Mol. BioZ. 33, 609. penman, S. (1966). J. Mol. BioZ. 17, 117. Penman, S., Smith, I. & Holtzman, E. (1966). Science, 164, 786. Penman, S., Vesco, C. & Penman, M. (1968). J. Mol. BioZ. 34, 49.

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Perry, R. P. (1962). Proc. Nat. Acad. Sci., Wash. 48, 2179. Petermann, M. L. & Pavlovec, A. (1966). B&him. biophys. Acta, 114, 264. Scherrer, K. & Darnell, J. E. (1962). Biophys. Biochem. Res. Comm. 11, 549. Scherrer, K., Marcaud, L., Zajdela, F., London, I. & Gros, F. (1966). Proc. Nat. Acad. Sci., Wash. 56, 1571. Wagner, E. K. & Ingram, V. (1966). Biochemistry, 5, 3019. Wagner, E. K., Katz, L. & Penman, S. (1967). Biochem. Biophys. Res. Comm. 28, 152. Warner, J. R., Soeiro, R., Birnboim, H. C., Girard, M. & Darnell, J. E. (1966). J. Mol. Biol. 19, 349. Weinberg, R. A., Loening, U., Willems, M. & Penman, S. (1967). Proc. Nat. Acad. Sk., Wash. 58, 1088. Willems, M., Wagner, E., Laing, R. & Penman, S. (1968). J. Mo2. Biol. 32, 211.