- Email: [email protected]
Intermolecular duplexes in heterogeneous nuclear RNA from HeLa cells
Cell, Vol.
10, 597-610,
April
1977,
Copyright
@ 1977 by MIT
Intermolecular Duplexes RNA from HeLa Cells Nina Fedoroff Carnegie Institution of Washington Department of Embryology 115 West University Parkway Baltimore, Maryland 21210 Peter K. Wellauer Swiss Institute for Experimental Cancer Ch. des Boveresses CH-1066 EPALINGES s/Lausanne Switzerland Randolph Wall Molecular Biology Institute University of California Los Angeles, California 90024
Research
Summary Rapidly sedimenting hnRNA complexes contain regions of stable intermolecular duplex. Disruption of such complexes, as judged by a reduction in sedimentation rate, requires conditions sufficient to denature the duplex regions. Rapidly sedimenting molecules reappear only when the complementary sequences reanneal-that is, the formation of such complexes is dependent upon time and the concentration of homologous RNA. These experiments lead us to the conclusion that rapidly sedimenting hnRNA complexes consist of two or more largely single-stranded RNA molecules held together by short duplex regions. Precisely such structures have been visualized in the electron microscope. Rapidly sedimenting fractions of native nuclear RNA from preparative sucrose gradients consist primarily of large, multimolecular complexes interconnected by duplex regions averaging 300 base pairs in length. Exposure of the RNA to severely denaturing conditions eliminates such complexes. Reannealing of the RNA reconstitutes complexes which are indistinguishable from those observed in preparations before denaturation. Introduction It has often been noted that exposure of eucaryotic heterogeneous nuclear RNA (hnRNA) to denaturing conditions effects a moderate to extreme reduction in its size, as judged by sedimentation rate or mobility on polyacrylamide gels (de Kloet, Mayo and Andrean, 1970; Mayo and de Kloet, 1971; Imaizumi, Diggelmann and Scherrer, 1973; Morrison and Busch, 1973; McKnight and Schimke, 1974; Macnaughton, Freeman and Bishop, 1974; Derman and Darnell, 1974; Spohr et al., 1976). Large, denaturable hnRNA complexes have been viewed variously
in Heterogeneous
Nuclear
as aggregates of unrelated molecules and as “nicked” or “processed” primary transcripts (de Kloet et al., 1970; Mayo and de Kloet, 1971; Derman and Darnell, 1974). Scant attention has been paid to defining the physical basis of hnRNA complex formation, a necessary prerequisite to understanding the origin and significance of such complexes. In this communication, we report experiments designed to test the hypothesis that hnRNA complexes are partial duplexes. It is apparent from several studies that hnRNA complexes are quite stable, readily surviving under conditions sufficient to dissociate the kinds of aggregates observed to form between bacterial RNAs (Hayes, Hayes and Guerin, 1966; Mayo and de Kloet, 1971; Morrison and Busch, 1973; Spohr et al., 1976). We have already reported that HeLa hnRNA, as it is extracted from the cell, contains intermolecular duplexes, as well as complementary sequences not in double-stranded form (Fedoroff and Wall, 1976). Here we establish a relationship between the sedimentation behavior of hnRNA and its content of intermolecular duplexes. We conclude that rapidly sedimenting hnRNA molecules are primarily multimers interconnected by short, rather stable, double-stranded regions. Such structures have been visualized in the electron microscope and predominate in rapidly sedimenting fractions of hnRNA. Results Evidence That Denaturable hnRNA Complexes Are Intermolecular Partial Duplexes We isolated HeLa hnRNA by two different methods. Method 1 is a modification of the procedure developed by Holmes and Bonner (1973), and involves lysis of nuclei in a strongly denaturing medium prior to phenol extraction of the nucleic acids (method 1). We have also employed the more widely used procedure developed by Penman (1966), in which the chromatin is disrupted with DNAase before phenol extraction (method 2). HeLa hnRNA extracted by method 1 contains a much higher fraction of rapidly sedimenting material than hnRNA extracted by method 2. This is apparent from the data given in Table 1. Before denaturation, more than half the hnRNA sample prepared by method 1 had an apparent sedimentation coefficient >65, and almost a quarter of it had an apparent sedimentation coefficient >lOO. The fraction of the RNA sedimenting with or ahead of 45s prerRNA varied between 60 and 90% in different preparations isolated by method 1. Less than 15% of the undenatured hnRNA sample prepared by method 2 sedimented between 65s and lOOS, and almost none had an apparent S value in excess of 100. RNA sedimenting ahead of 455 pre-rRNA has a
Cell 598
Table
1. Duplex
Content
of hnRNA
Differing
in Sedimentation
Properties % Duplex’
RNA Extraction Method
Method
1
2
Procedure
Sedimentation
Range”
% Total
MateriaP
Native
Denatured
65S-100s
32
5.0
1.4
>I00
23
5.7
1.7
32S-45s
37
1.4
1.2
5OS-65s
9
4.6
1.6
13
4.9
1.4
65S-100s a Calculated with respect to rRNA standards. b Fraction of total labeled material in the indicated S value range. r Determined by RNAasedigestion without (native) or with (denatured) The duplex content was assayed by cellulose-ethanol chromatography. hnRNA was extracted from HeLa cells labeled for 2 hr with JH-uridine
higher duplex content than RNA sedimenting more slowly than 45s pre-rRNA (Table 1). Preliminary denaturation of RNA prepared by either method yields material sedimenting predominantly between the 28s and 45s rRNA markers on nondenaturing gradients (see Figure 2 below for an example). Preliminary denaturation also reduces the duplex content to ~2% (Table 1). These results suggest that double-stranded regions stabilize large complexes. Rapidly sedimenting hnRNA complexes dissociate concomitantly with melting of the duplex regions. The concentration of dimethyl sulfoxide (MeSO) required to denature RNA at room temperature is a function of its melting temperature (Strauss, Kelly and Sinsheimer, 1968). Samples of rapidly sedimenting hnRNA were therefore exposed to increasing concentrations of MeSO at 25°C. Parallel aliquots were withdrawn for analysis of sedimentation behavior and assay of duplex content. The results of this experiment are shown in Figure 1. The sedimentation data are represented, for convenience, by the fraction of material sedimenting with or ahead of 45s pre-rRNA. There is a concomitant decrease in duplex content and sedimentation rate between 60 and 80% MeSO. This is in the range expected for double-stranded RNA and higher than necessary to denature either single-stranded RNA or double-stranded DNA (Helmkamp and Ts’o, 1961; Strauss et al., 1968). Rapidly sedimenting hnRNA complexes reform under conditions favoring renaturation of intermolecular duplexes. Highly labeled hnRNA was mixed either with excess unlabeled hnRNA or unlabeled E. coli RNA and denatured with MeSO. Each sample was concentrated by ethanol precipitation, resuspended in a buffer of high ionic strength and annealed at 55°C. Aliquots were withdrawn at 2, 7.5 and 20 min for analysis on nondenaturing sucrose gradients. The results are shown in Figure 2. There is a marked shift in the sedimentation profile with time in samples containing a high hnRNA concen-
preliminary
heating
and fractionated
in 0.01 M Tris (pH 7.4). 0.001 M EDTA at 100°C. on nondenaturing
sucrose
gradients.
80
I 60 2 u rn : Al 4o
I
I
20 40 % OIMETHY
I
L
$
1
60 80 SULF OXIDE
Figure 1. Effect of Increasing Dimethyl on the Sedimentation Rate and Duplex
Sulfoxide Concentration Content of hnRNA
Nuclear RNA was purified by method 1 from HeLa cells labeled for 2 hr with JH-uridine in the presence of 0.04 pg/ml actinomycin D, then layered on a nondenaturing 15-30% sucrose gradient and centrifuged in an SW56 rotor at 56,000 rpm for 75 min at 25°C. Material which pelleted was dissolved in 0.01 M Tris (pH 7.4). 0.001 M EDTA. ethanol-precipitated and redissolved in 0.1 M NaCI. 0.05 M Tris (pH 6.85), 0.001 M EDTA. Aliquots were diluted IO fold into ice-cold aqueous Me&O to give the indicated final concentrations and incubated at 25°C for 10 min. Aliquots were withdrawn for determination of duplex content by celluloseethanol chromatography after RNAse digestion (see Experimental Procedures) and for sedimentation on nondenaturing sucrose gradients. The results are expressed as the fraction of material sedimenting with or ahead of the 32P-45S pre-rRNA marker included in each gradient (A-A) and as the fraction of total radioactivity in a ribonuclease digest eluting at the position of duplex RNA from cellulose (O---O).
Duplexes 599
in hnRNA
tration. By 2 min of incubation, the fraction of material sedimenting with or ahead of 4% prerRNA has almost doubled. By 20 min, almost 80% of the RNA sediments with or ahead of 45s prerRNA. No such shift was observed in control samples containing unlabeled E. coli RNA. After 20 min of incubation, there is no detectable shift in the sedimentation profile (Figure 2D, broken line). (The total RNA concentration was lower by a factor of 5 in the control containing E. coli RNA. Hence the terminal time point represents a 4 min incubation at an RNA concentration equivalent to that in the experimental sample.) Since the annealing times were the same in both experimental and control samples, the shift in sedimentation coefficient in the former cannot be attributed to intramolecular renaturation (Schmid, Manning and Davidson, 1975). The shift in sedimentation profile requires sequences present in hnRNA, but not in E. coli RNA, ruling out nonspecific aggregate formation. Since about half the sequences that can anneal in hnRNA would have annealed after incubation for 20 min at the high hnRNA concentration (Fedoroff and Wall, 1976), it appears probable that the formation of rapidly sedimenting complexes is consequent on annealing of complementary sequences. Since only a small fraction of the hnRNA becomes double-stranded, while the sedimentation profile of virtually the entire population is affected, it follows that the complexes consist of largely singlestranded molecules held together by short duplex regions. There is rapid formation of complexes sedimenting much faster than would be expected for dimers. This suggests that a given hnRNA molecule contains more than one short sequence capable of finding a complementary sequence on another molecule. Some Properties Thermal melting Figure 2. The Sedimentation
FRACTION
of the Duplex Regions in hnRNA curves for the intermolecular du-
Effect of Reannealing Properties
Denatured
hnRNA
on
Its
Nuclear RNA was prepared as described in the legend to Figure 1, except that all the material sedimenting with or ahead of 45s prerRNA was pooled during the initial gradient purification. Aliquots of labeled hnRNA (5 x 10’ cpm. 1.6 x lo6 cpmlpg) were mixed with 20 pg of unlabeled hnRNA in 0.01 M Tris (pH 7.4), 0.001 M EDTA, adjusted to 75% Me&O and heated at 55°C for 3 min. The RNA was then ethanol-precipitated, dissolved in HzO, adjusted to 0.5 mg/ml RNA in 0.4 M NaCI, 0.2 M Tris (pH 6.85),0.004 M EDTA. 10% formamide, and incubated for (A) 0 min, (6) 2 min. (C) 7.5 min and (D) 20 min at 55°C (04). Controls were treated identically, except that unlabeled hnRNA was replaced by unlabeled E. coli rRNA. and the annealing reaction was carried out at a final RNA concentration of 0.1 mg/ml. Only the 20 min time point is shown (D) (0- - -0). The samples were layered on nondenaturing lo30% sucrose gradients and centrifuged in an SW41 rotor at 40,000 rpm for 130 min at 25°C. 32P-45S pre-rRNA was included in each gradient as an internal marker. Sedimentation is from right to left.
Cell 600
plexes are shown in Figure 3. The T, values obtained were 77°C in 0.1 x SSC and 95°C in 1 x SSC. To follow the dissociation only of intermolecular duplexes, undenatured hnRNA was heated to the indicated temperatures, chilled and RNAase-digested. Double-stranded material was then assayed by cellulose-ethanol chromatography (see Experimental Procedures). This permits intramolecular duplexes to reform and excludes them from the measurement. Of the initial duplex population, 25-30% remains double-stranded in such an experiment. Brief incubation at the low hnRNA concentration used did not further increase this value (data not shown). The melting curves are quite sharp, and the T, values are virtually identical to those obtained with double-stranded RNA prepared by self-annealing in vitro transcripts of denatured bacteriophage T4 DNA. This duplex RNA gave T, values of 79°C in 0.1 x SSC and 95°C in 1 x SSC. A similar duplex RNA standard prepared using adenovirus type 2 DNA as template gave T,,, values of 86°C and 101°C at the two salt concentrations, respectively. The difference in the melting temperatures of the RNA standards reflects the difference in the GC content of the DNA templates used (Billeter, Weissmann and Warner, 1966). The GC content of T4 DNA is 34% and that of adenovirus DNA is 55%. Since the melting curves for the intermolecular duplexes in hnRNA are relatively
-I B
I
I
70
90
TEMPERATURE Figure 3. Thermal hnRNA
Melting
Curves
,
I
70
90
(“C)
of Intermolecular
Duplexes
in
Nuclear RNA was prepared as described in the legend to Figure 1, but not fractionated. Total, undenatured hnRNA was heated in 0.1 x SSC (A) or 1 x SSC (B), diluted with 0.2 M NaCI. 0.1 M Tris (pH 6.65), 0.002 M EDTA, RNAase-digested and subjected to cellulose-ethanol chromatography. The preparation used in this experiment contained 5% double-stranded RNA (100%). Intact hnRNA was used in this experiment to permit reformation of intramolecular duplexes before RNAase digestion, excluding them from the measurement. Roughly one quarter of the total double-stranded RNA behaved as intramolecular duplexes.
sharp, the low T, probably reflects a low GC content for the duplex population. The sequences which are double-stranded in undenatured hnRNA appear to be the most abundant complementary sequences in hnRNA. Doublestranded RNA was isolated by cellulose-ethanol chromatography from undenatured hnRNA prepared by method 1 and digested with RNAase. The recovered double-stranded material was denatured and reannealed to a Rot of 0.15 M.sec (normalized to the initial hnRNA concentration). The sample was then separated into double-stranded and single-stranded fractions by cellulose-ethanol chromatography without RNAase digestion. The fraction remaining single-stranded was further annealed to a Rot of 15 M set and refractionated. Of the total complementary sequences in hnRNA, roughly 17% are double-stranded at a Rot of 0.15 M.sec (Fedoroff and Wall, 1976), as compared with 38% for the renatured duplexes. At the higher Rot of 15 M.sec, roughly half the total complementary sequence population is double-stranded (Fedoroff and Wall, 1976), while 78% of isolated duplexes have reannealed. Although these experiments are not completely comparable, since isolated, reannealed double-stranded RNA was fractionated without RNAase treatment, they nonetheless suggest that complementary sequences which are double-stranded in hnRNA as it is isolated from the cell are those that are most abundant in the total complementary sequence population. The complementary sequences that are doublestranded in undenatured hnRNA are almost exclusively transcripts of highly reiterated DNA. This was determined by hybridizing the Rot-fractionated duplexes obtained in the preceding experiment to a 300,000 fold excess of human placental DNA. The three fractions whose hybridization curves are shown in Figure 4 represent material reannealing at values of Rot up to 0.15 M.sec, at values of Rot between 0.15 M.sec and 15 M.sec, and at values of Rot >15 M.sec. Hybridization was assayed by the development of RNAase resistance. Reassociation of the RNA was minimized by the choice of conditions and monitored by the inclusion of controls lacking DNA, but containing RNA. The RNAase resistance developed in control samples is plotted in the lower curves of Figure 4 as a function of the Cot attained in DNA-containing experimental samples incubated for the same length of time. The upper curves show the RNAase resistance in experimental samples as a function of Cot. About 90% of the observed hybridization occurs at Cot values below 10 Msec. Only the most slowly annealing fraction contains material which hybridizes at higher Cot values (Figure 4C). Hence the complementary sequences that are duplexed in native hnRNA are
Duplexes 601
in hnRNA
10-1
10-Z
10-l
1
10
102
IO'
CO'
Figure 4. Hybridization to DNA of Rot-Fractionated Derived from Undenatured hnRNA
Duplex
RNA
Nuclear RNA, labeled as described in the legend to Figure 1, and sedimenting with or ahead of 455 RNA in a preparative, nondenaturing sucrose gradient, was digested with RNAase. The duplex RNA was purified by cellulose-ethanol chromatography. The double-stranded RNA was heat-denatured and reannealed to a Rot of 0.15 M’sec (based on the initial hnRNA concentration), then separated into single- and double-stranded fractions by celluloseethanol chromatography without RNAase digestion. The singlestranded fraction was further annealed to a Rot of 15 M.sec, and the fractionation was repeated. Aliquots were then hybridized to a 300,000 fold excess of sheared human placental DNA in phosphate buffer at 60°C and assayed for ribonuclease resistance developed in the presence (upper curves) or absence (lower curves) of DNA. Control values of ribonuclease resistance are plotted as a function of the Cot attained in experimental tubes incubated for the same length of time. (A) hnRNA reannealing at Rot%O.lS M.sec; (8) hnRNA reannealing at 0.15sRotG15 M.sec; (C) hnRNA reannealing at Rot>15 M’sec.
transcribed from highly reiterated DNA. This is, once again, by contrast with the total complementary sequence population in hnRNA, which contains transcripts of DNA sequences present at high, moderate and low reiteration frequences (Fedoroff and Wall, 1976). Electron Microscopic Analysis of hnRNA The basic difficulties in analyzing hnRNA by electron microscopy proved to be the very large size of the hnRNA complexes and the varied array of structures. hnRNA spread from 30% formamide/ M urea showed extensive secondary structure, as well
as intermolecular associations. Many molecules were almost completely collapsed into loops and hairpins. Increasing concentrations of formamide/ urea gradually eliminated secondary structure. Molecules apread from 90% formamide/ M urea had little secondary structure, yet most molecules remained in multimolecular complexes. The hnRNA preparation used here was purified by method 2. The RNA sedimenting ahead of 45s pre-rRNA after a single round of centrifugation was found to contain structures characteristic of rRNA (Wellauer and Dawid, 1973). It was therefore repurified on a second sucrose gradient, and molecules having an apparent S value range of 65-100 were pooled and used for electron microscopy. Electron micrographs of undenatured hnRNA molecules are shown in Figures 5 and 6. RNA was spread from 90% formamide/4.5 M urea. The complexes shown contain three or four distinct singlestranded RNA molecules connected by short duplex regions. Each complex contains at least double-stranded regions. Although the duplex regions are short and strikingly uniform in length, fairly long ones are occasionally observed (Figure 5). In measuring duplex lengths, we have omitted from consideration crossover points such as those indicated by broken arrows in Figure 5, since these cannot reliably be identified as duplexes. The single-strand distance separating two duplex regions is highly variable, as are the lengths of the single-stranded tails emerging from a duplex region. The molecules shown here are particularly well spread out. Even when spread from 90% formamide/4.5 M urea, most complexes are sufficiently tangled to preclude enumeration of the molecules with certainty. Because of this, we cannot make meaningful quantitative statements either about the frequency of such complexes or about the number of molecules per complex. Qualitatively, however, we can state that most of the hnRNA mass is in such complexes when the RNA is spread from 90% formamide/4.5 M urea. Preliminary denaturation eliminates large complexes. Occasional dimers remain. Such structures were not included in length measurements. A histogram showing the lengths of denatured hnRNA molecules is shown in Figure 7. The calculated number average length is about 4100 nucleotides, and the weight average length is 7100 nucleotides. An aliquot of the denatured preparation used to generate the length histogram in Figure 7 was renatured for 10 min at 55°C in 0.4 M NaCl, 0.2 M Tris (pH 6.85), 0.004 M EDTA, 10% formamide and at a concentration of about 150 pg/ml before spreading. It is essentially indistinguishable from the native preparation. A relatively well spread complex from the renatured preparation is shown in Figure
Cell 602
Figure
5. Electron
Micrograph
of an hnRNA
Complex
Spread
from
90% Formamide/4.5
M Urea
HeLa hnRNA was extracted by method 2 from cells treated with actinomycin D to suppress rRNA synthesis. Material sedimenting ahead of 45s rRNA on a nondenaturing sucrose gradient was pooled and rerun on a second sucrose gradient. Material running in the 65S-100s region was pooled, ethanol-precipitated twice and then redissolved in 10 mM Tris (pH 7.5) for spreading. The tracing (b) shows our interpretation of the photograph (a). Single-stranded RNA is drawn as a thin line and double-stranded RNA as a thick line. Single-stranded ends are indicated by solid arrows and intermolecular duplexes by open arrows. An additional region which may comprise a short duplex is indicated by the broken arrow. Such structures were observed frequently, but were omitted from measurements of duplex length (Table 2), since they are not distinguishable in the electron micrographs from fortuitous crossing-over of free RNA molecules. Bar = 0.1 pm.
Duplexes 603
in hnRNA
b Figure
6. Electron
The details
Micrograph
of hnRNA
preparation
of an hnRNA
Molecule
are given
in Figure
Spread
from
90%
FormamidW4.5
M Urea
5. Bar = Cl.3 pm.
8. It should be noted that renaturation reconstitutes complexes containing more than two molecules, as expected from the results of the experiment represented in Figure 2. In Figure 9, we show the different kinds of duplex structures encountered in native hnRNA. Since the preparations were spread from 90% formamide/
urea, these represent the most stable inter- and intramolecular duplexes in the RNA. These structures include hairpins and loops (a and b), forks and duplexes with four single-stranded tails of various lengths (c and d). The duplex lengths were measured for two broad categories, intra- and intermolecular duplexes. Included in the intramolec-
Cell 604
LENGTH
Figure Cells
7. Length
Distribution
(pm)
Histogram
of hnRNA
from
HeLa
The RNA was denatured by boiling in 10 mM Tris (pi-i 7.5) for 2 min and spread immediately from 90% formamide/4.5 M urea. The number average length of 630 molecules is 1.29 pm, or about 4100 nucleotides. The weight average length is 2.24 pm. corresponding to about 7100 nucleotides. The length was determined by comparison with E. coli 23s rRNA. which was spread separately under similar conditions. The few small complexes that were still present after denaturation were omitted from these measurements.
ular category were all loops and hairpins. Other structures were considered intermolecular. This distinction between intra- and intermolecular is somewhat arbitrary in the case of hairpins, since discontinuities in primary structure within such regions cannot be distinguished in the electron microscope. The lengths of intra- and intermolecular duplexes, respectively, in native hnRNA spread from 90% formamide/ urea are given in Table 2. Both have an average length of about 0.1 p, which corresponds to about 300 base pairs. The lengths of intermolecular duplexes in native hnRNA spread from 30% formamide/l.5 M urea, and in denatured and reannealed hnRNA spread from 90% formamide/4.5 M urea are also given in Table 2. The average length is the same in all cases. The length of double-stranded RNA isolated after ribonuclease digestion was determined by polyacrylamide gel electrophoresis to be in the range of 50-200 base pairs (data not shown). Assuming that the RNAaseresistant duplexes are representative of those observed by electron microscopy, this result indicates the presence of RNAase-sensitive mismatches within the double-stranded regions. Discussion We have been quite impressed with the difficulty of obtaining hnRNA completely free of partially duplexed structures. hnRNA samples exposed to 90% MeSO or heated to 100°C in 10 mM Tris (pH 7.5), then ethanol precipitated before spreading for electron microscopy, contained a few dimers. Surprisingly, even an hnRNA sample boiled in water
immediately before spreading contained dimers. Such residual partial duplexes might represent regions which have not denatured, either because of their base composition or because they are crosslinked in some way. Alternatively, and perhaps more probably, such duplexes might result from the renaturation of some of the complementary sequences on different RNA molecules. Reannealing of complementary sequences is observed at Rot values between 10e3 and lO-2 M.sec (Fedoroff and Wall, 1976). Significant reassociation at room temperature might easily be expected within a period of a few minutes at the formamide and urea concentrations used during RNA spreading for electron microscopy. We believe that the complementary sequences studied here are not generally double-stranded in the cell. This contention rests on the observation that hnRNA prepared by two different methods from the same cells differs markedly in duplex content. Correlated with the difference in duplex content is a marked difference in sedimentation behavior-that is, the higher the content of intermolecular duplexes, the more rapidly does the RNA sediment in a sucrose gradient. Pronounced variation in the sedimentation profiles of undenatured hnRNA is the rule rather than the exception in the published literature (Bramwell and Harris 1967; Georgiev et al., 1972; Morrison and Busch, 1973; Derman and Darnell, 1974; Macnaughton et al., 1974). Perhaps the basis of this variation is the extent to which complementary sequences normally not duplexed in the cell are able to anneal during isolation and manipulation of the hnRNA. Supporting evidence that similar sequences can be either single- or double-stranded in hnRNA as it is isolated comes from the following observations. Ryskov et al. (1975) reported that when labeled, double-stranded RNA isolated from murine ascites hnRNA was denatured and reannealed, its renaturation rate could be accelerated by the addition of unlabeled and undenatured hnRNA. This means that undenatured hnRNA contains, in singlestranded configuration, sequences which can also be present in double-stranded form. There are several large RNAase Tl oligonucleotides highly characteristic of duplex regions in HeLa hnRNA (H. D. Robertson, W. Jelinek and E. Dickson, personal communication). A smaller fraction of these sequences is cleaved by the double-strand-specific RNAase III in total hnRNA than in isolated hnRNA duplexes (H. D. Robertson et al., personal communication). In addition, hnRNA recovered from DNARNA hybrids obtained at low values of Cot gives the Tl oligonucleotide pattern characteristic of double-stranded regions. However, much more of the hnRNA is recovered from such hybrids than is present in the form of double-stranded RNA (R. Evans,
Duplexes 605
in hnRNA
cI 7
-
-? I
I b
Figure
6. Electron
Micrograph
of a Complex
in a Denatured
and Reannealed
hnRNA
Sample
Spread
A sample of hnRNA, prepared as described in Figure 5, was denatured by heating to 100°C in 10 mM was adjusted to 0.4 M NaCI, 0.2 M Tris (pfi 6.65), 0.004 M EDTA, 10% formamide, and the sample was was precipitated with ethanol, redissolved in IO mM Tris (pfi 7.5) and spread from 90% formamide/4.5 at least six individual molecules joined by five duplex regions (open arrows). Single-stranded tails secondary structure is also seen in this particular complex. The insert shows a small dimeric molecule of hnRNA. Bar = 0.3 &rn.
from
90% Formamide/4.5
M Urea
Tris (pfi 7.5). The salt concentration annealed for 10 min at 55°C. The RNA M urea. The complex shown contains are indicated by solid arrows. Some from the same renatured preparation
Cell 606
Figure
9. Electron
Micrographs
and Tracings
of Various
Types
of Duplex
Structures
Observed
in hnRNA
(a) photographs of intramolecular duplexes. These are double-stranded structures, without or with a terminal single-stranded loop, and with two single-stranded tails. (c) photographs of intermolecular duplexes. These include, from left to right, forked structures, forked structures with one or two short whiskers and structures with four long single-stranded tails. In (e) and (f), intramolecular (double arrows) and intermolecular (single arrows) duplexes are shown for comparison in the same molecule. Note that the two types of duplexes are of similar length. The tracings (b and d) show our interpretation of the various structures. Single-stranded RNA is represented by thin lines and double-stranded RNA by hatched bars to indicate hydrogen bonding. Long single-stranded tails are indicated by dashes. The bar of 0.1 pm corresponds to about 300 bases/base pairs.
Duplexes 607
Table
in hnRNA
2. Size of Duplex
Regions
in HeLa
Cell hnRNA Spreading Conditions
L, + SD pm (Number Molecules)”
of
Number Pairs”
RNA
Type of duplex
Native
Intermolecular
30% formamide, 1.5 M urea
0.091
+ 0.036
(146)
-290
Native
Intermolecular
90% formamide. 4.5 M urea
0.097
+ 0.049
(112)
-310
Reannealed
Intermolecular
90% formamide. 4.5 M urea
0.090
+ 0.026
(156)
-290
Native
Intramolecular’
90% formamide, 4.5 M urea
0.083
f 0.032
(138)
-260
of Nucleotide
a L, = number average length. ’ E. coli 23s rRNA (3070 nucleotides; Nikolaev, Schlessinger and Wellauer, 1974) spread under similar conditions has an average linear density of 1 .l x lo6 daltons per pm. The single-strand molecular weight of duplexes in hnRNA was obtained by multiplying length with linear density of E. coli rRNA. Duplex and single-stranded RNA have very similar linear densities (Wellauer and Dawid, 1973) under the spreading conditions used. The number of nucleotide pairs was determined by dividing the molecular weight of duplexes by the average molecular weight per nucleotide. ’ In view of the spreading conditions used, the intramolecular duplexes measured here represent the most stable ones in hnRNA. The two types of secondary structure features that were measured are fully double-stranded structures and structures with a double-stranded stem and a loop at the end (see Figure 9); only the stem was measured in structures with loops.
personal communication). Once again, these observations suggest that not all of this simple complementary sequence population is doublestranded in the hnRNA as it is extracted from the cell. There is ample reason to believe that the environment to which the RNA is exposed during extraction promotes the annealing of complementary sequences. In particular, it has recently been shown that annealing reactions are markedly accelerated in a two-phase, phenol-aqueous system (Kohne, Levinson and Byers, 1977). There are two quite well documented examples of highly specific RNA annealing reactions which occur during extractions involving phenol. These are the stable partial duplexes formed by 28s rRNAs and pre-rRNAs of Xenopus laevis tissue culture cells (Hagenbuchle, Schibler and Wyler, 1975) and the double-stranded structures formed by complementary, replicating bacteriophage RNAs during isolation (Weissmann, Feix and Slor, 1968). But in both these instances, and probably in the case of hnRNA, where we have obtained preparations consistently differing in duplex content by two methods both involving phenol extraction, the explanation is undoubtedly more complex than merely phenol-mediated acceleration of annealing. Such parameters as local concentrations of complementary sequences at the time of deproteinization may well be important. Simple reconstruction experiments, where they have been tried, have failed (Weissmann et al., 1968; Hagenbuchle et al., 1975). It has been suggested that denaturable hnRNA complexes are ‘nicked” or “processed” primary transcripts (Derman and Darnell, 1974). The implication here is that complexes contain contiguous transcripts whose summed length approximates
that of the transcription unit. In the preceding discussion, we have argued that many, if not all, of the denaturable complexes are formed during isolation and manipulation of the RNA. This could be true, yet not preclude formation of highly specific complexes containing only contiguous transcripts. If such specific complexes did form during isolation of the RNA, their sequence composition would be very different from that of complexes formed during in vitro reannealing of denatured hnRNA. This follows from the fact that there are many copies of relatively few complementary sequences. H. D. Robertson et al. (personal communication) have estimated that the total complexity of the duplex regions in HeLa hnRNA is < 1000 nucleotides. By kinetic means, we have estimated that the simplest complementary sequence component in hnRNA has a complexity of roughly 1500 base pairs (Fedoroff and Wall, 1976). It is therefore far more probable that upon renaturation, a given sequence anneals to a complementary region on a different molecule from the one it was initially joined to, than that it finds its initial complement. Despite this randomization, our blunt tools find the native and renatured preparations indistinguishable. Even in the electron microscope, they appear to contain the same kinds of multimolecular networks held together by the same range of duplex structures of the same length. This means either that all complexes are random partial duplexes or that specific complexes cannot be distinguished structurally from random complexes. The relationship between the sequences we have studied here and the “hairpin” RNA sequences described by other investigators is not clear (Jelinek and Darnell, 1972; Ryskov et al., 1973). Unless care is taken to distinguish kinetically between intramo-
Cell 606
lecular and rapid intermolecular duplex formation, any given population of double-stranded RNA isolated from hnRNA would tend to contain both kinds of sequences. There is no inherent reason, however, that the two populations need have a different sequence content. Sequences in intramolecular duplexes could be transcribed from a subset of the same family of highly reiterated sequences in DNA as those in intermolecular duplexes. Some members of this family of sequences might occur as inverted repetitions in close juxtaposition giving rise to “hairpinned” transcripts. In summary, our experiments show that heterogeneous nuclear RNA readily forms networks of molecules interconnected by double-stranded regions averaging 300 base pairs in length. This property of hnRNA renders suspect length measurements made under any but the most rigorously denaturing conditions (Spohr et al., 1976) and may preclude resolution of questions about the length of transcription units from analyses of purified transcripts. The complementary sequences which are duplexed in undenatured hnRNA are quite abundant, have a low overall sequence complexity and form stable duplexes. We wish here to focus attention on their simplicity and ubiquity. These properties suffice to make such sequences good candidates for cleavage sites in the reductive “processing” that has been postulated to occur in the synthesisof eucaryotic mRNAs (Darnell, Jelinek and Malloy, 1973). We believe it important, however, to consider the possibility that these simple, abundant sequences find their significance at other levels of genetic organization and expression. They could, at one extreme, reflect the molecular architecture of the genome and have no function as transcripts. At the other extreme, they could have an autonomous function in nuclear RNA metabolism. Theoretical constructs postulating specific roles for transcripts of reiterated DNA sequences have appeared recently (Robertson and Dickson, 1974; Davidson, Klein and Britten, 1977). Experimental
Procedures
Cell Labeling and Isolation of Nuclei HeLa cells growing in Eagle’s medium were concentrated to 4 x lO”/ml and incubated with 0.04 pg/ml actinomycin D for 25-30 min before labeling. 3H-uridine (5 mCi/lO ml cells, 29.2 Ci/mmole) was added, and incubation continued for 2 hr (Derman and Darnell, 1974). Labeling was terminated by pouring cells over frozen, crushed, phosphatebuffered saline. Unlabeled hnRNA was extracted from cells incubated in the presence of 0.04 pg/ml actinomycin D for 3 hr. Nuclei were prepared from labeled cells as described by Penman (1966). Unlabeled cells were concentrated by centrifugation,
resuspended at 10B/ml in cold 0.32 M sucrose (Schwarz/Mann, RNAase-free), 3.0 mM MgCI,, 500 pglml heparin (Riker Laboratories), and lysed in a Kontes Cell Disruption Bomb. The lysate was adjusted to 500 pg/ml spermidine and centrifuged at 2000 x g for 5 min. Nuclei were then detergentwashed as described by Penman (1966). hnRNA Extraction-Method 1 Nuclei were taken up in 1 ml/l-2 x 10’ starting cells of cold nuclear lysis solution containing 7 M urea (Schwarz/Mann, Ultrapure), 0.35 M NaCI, 0.01 M Tris (pH 7.6), 0.001 M EDTA, 2% Sarkosyl (M Chemical Co.), and 5% phenol (Mallinckrodt, giltlabel). The resulting viscous solution was gently homogenized in a Dounce homogenizer using a loose-fitting pestle. 1 vol of a 3:l mixture of phenol and chloroform containing 5% isoamyl alcohol was added, and the solution was brought quickly to 3040°C with shaking. Phases were separated by low speed centrifugation at room temperature. The phenol phase was reextracted with 0.5 vol of the nuclear lysis mixture, and the pooled aqueous phases were reextracted with phenol mixture, followed by dialysis against cold 0.1 M Na acetate (pH 7.0) in diethyl pyrocarbonate-treated dialysis tubing. The solution was adjusted to 0.01 M MgCI,and 10 pg/ml iodoacetate-treated DNAase (Zimmerman and Sandeen, 1966), incubated at 37°C for 15 min and phenol-extracted as above, following addition of EDTA to 0.01 M and sodium dodecylsulfate to 0.5%. This procedure is a modification of that described by Holmes and Bonner (1973). Ethanolprecipitated RNA was collected by centrifugation and washed with 3.0 M Na acetate (pH 6.0) to remove residual DNA fragments (Macnaughton et al., 1974), then with 0.2 M Na acetate (pH 6.0), 75% ethanol. hnRNA Extraction-Method 2 Nuclei were digested with iodoacetate-treated DNAase, and RNA was extracted as described Penman (1966).
by
Sucrose Density Gradient Sedimentation Nondenaturing gradients were 15-30% sucrose in a buffer consisting of 0.1 M NaCI, 0.01 M Tris (pH 7.4), 0.001 M EDTA and 0.1% SDS. After denaturation in MeSO, samples were diluted at least 3 fold with 0.01 M Tris (pH 7.4), 0.001 M EDTA before layering on such a gradient. Annealing RNA was denatured by heating to 100°C for 3 min in 0.01 M Tris (pH 7.4), 0.001 M EDTA. Annealing was carried out in 0.4 M NaCI, 0.2 M Tris (pH 6.85), 0.004 M EDTA, 10% formamide in sealed capillaries at 55°C. The extent of annealing was monitored
Duplexes 609
in hnRNA
by cellulose-ethanol RNAase digestion.
chromatography
following
RNAase Digestion RNA was digested with 0.5 pg Tl RNAase (Calbiochem) and 1 unit of T2 RNAase (Calbiochem) per 20 pg of RNA in 0.2 M NaCI, 0.1 M Tris (pH 6.85), 0.002 M EDTA. The RNA concentration was maintained at 50 pg/ml by addition of E. coli rRNA when necessary. RNAase-resistant material was measured by applying the digest directly to a celluloseethanol column. DNA-RNA Hybridization Isolated double-stranded hnRNA was hybridized to human placental DNA (a gift from Dr. M. Shoyab), sonicated to an average length of 500 nucleotides, in Na phosphate buffer (pH 6.8). Hybridization was carried out in 0.03 or 0.2 M phosphate buffer, and equivalent Cot values were calculated as described by Britten, Graham and Neufeld (1974). Hybrids and controls lacking DNA were supplemented with 20 pg E. coli rRNA, and digested with 1 pg of Tl RNAase and 1 unit of T2 RNAase in 0.3 M NaCI, 0.03 M Na citrate for 30-60 min at 37°C. Samples were precipitated with 5% CCICOOH, and precipitates were collected on millipore filters for liquid scintillation spectrometry. Cellulose-Ethanol Chromatography Chromatography on cellulose (Sigmacell Type 38, Sigma) was performed as described by Franklin (1966). Columns were equilibrated with a buffer consisting of 35% absolute ethanol and 65% 0.1 M NaCI, 0.05 M Tris (pH 6.85), 0.001 M EDTA; samples were applied in the same buffer. The columns were washed with 35% ethanol buffer, then with the same buffer containing 15% ethanol. Doublestranded RNA was eluted with buffer alone (0% ethanol). Analytical columns were prepared in siliconized Pasteur pipets. RNAase digests in 0.2 M NaCI, 0.1 M Tris (pH 6.85), 0.002 M EDTA were applied to columns after dilution with 1 vol of 70% ethanol. Fractions were collected directly into vials, mixed with Triton X-100 scintillation fluid and counted. Mono- and oligonucleotides do not bind to the columns in the 35% ethanol sample application buffer. Single-stranded RNA elutes in 15% ethanol, and double-stranded RNA elutes in buffer alone. On a preparative scale, double-stranded RNA was isolated from hnRNA by RNAase digestion as described above, followed by passage through two successive cellulose columns (approximately 1 ml packed bed volume per pg double-stranded RNA). In Vitro Synthesis of RNA RNA complementary to T4 and adenovirus type 2 DNAs was synthesized in a reaction mixture con-
taining 0.04 M Tris (pH 7.9), 0.05 M KCI, 0.01 M MgCI,, 0.1 mM dithiothreitol, 0.4 mM ATP and GTP, 0.13 mM CTP and UTP, 0.01 mM 3H-CTP (Schwarz/ Mann, 25 CVmmole), 0.01 mM 3H-UTP (SchwartzMann, 18.6 Ci/mmole), 110 pg/ml heat-denatured T4 DNA (a gift from Dr. D. Kaplan) or 100 pglml heat-denatured adenovirus type 2 DNA, and 120 pglml E. coli RNA polymerase (400 units per mg; a gift from Dr. W. Mangel). At the end of 2 hr at 37”C, iodoacetate-treated DNAase was added to 180 pgl ml, and incubation at 37°C continued for 20 min. The reaction mixture was phenol-extracted, and the RNA was freed from nucleotides by celluloseethanol chromatography. Double-stranded RNA was prepared by self-annealing, RNAase digestion and cellulose-ethanol chromatography as described above. Electron Microscopy RNA was spread for electron microscopy as described in detail earlier (Wellauer and Dawid, 1973, 1974). Formamide/urea solutions of varying denaturing power were obtained by appropriate dilution of stock solution of 5 M urea in 99% formamide (Fisher certified, used without further purification). The concentration of the remaining components in the final spreading solution were: 0.5 pglml of RNA, 30 mM Tris (pH 8.5), -1 mM EDTA and 30 wg/ ml of cytochrome c. The hypophase was distilled water. Pictures were taken in a Hitachi HU-11 El electron microscope at a magnification of 14,000X. For each set of pictures, the magnification of the instrument was calibrated with a carbon replica of a diffraction grating (2160 lines per mm). Negatives were enlarged in a projector to a final magnification of 300,000X and measured with a graphics calculator (Numonics). Acknowledgments We gratefully acknowledge the excellent technical assistance of Kathleen Toth. This work was supported by grants from the NIH. the California Institute for Cancer Research, and the Swiss National Science Foundation. Some of this work was performed during the tenure of a postdoctoral fellowship from the Damon Runyan-Walter Winchell Fund for Cancer Research (N. F.). Received
October
12, 1976; revised
December
17, 1976
References Billeter, M. A., Weissmann. C. and Warner, R. C. (1966). Replication of viral ribonucleic acid: IX. Properties of double-stranded RNA from Escherichia coli infected with bacteriophage MSZ. J. Mol. Biol. 17, 145-173. Bramwell, M. E. and Harris, H. (1967). The origin of the polydispersity in sedimentation patterns of rapidly labelled nuclear ribonucleic acid. Biochem. J. 103, 816-830. Britten, R. J., Graham, D. E. and Neufeld, B. R. (1974). Analysis of repeating DNA sequences by reassociation. In Methods in Enzy-
Cell 610
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29E, L. Grossman Press), p. 363.
and
K. Moldave,
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York:
Darnell, J. E., Jelinek, W. R. and Molloy, G. R. (1973). Biogenesis of mRNA: genetic regulation in mammalian cells. Science 787, 1215-1221. Davidson, E. M., Klein, W. H. and Britten, R. J. (1977). Sequence organization in animal DNA and a speculation on hnRNA as a coordinate regulatory transcript. Dev. Biol.. in press. de Kloet. gregation presence 454-460.
S. R., Mayo, V. S. and Andrean, B. A. G. (1970). Disagof giant “DNA-like RNA” of yeast by denaturation in the of formaldehyde. Biochem. Biophys. Res. Commun. 40,
Penman, S. (1966). RNA metabolism Mol. Biol. 77, 117-130.
in the HeLa cell nucleus.
Robertson, H. D. and Dickson, E. (1974). control of gene expression. Brookhaven
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RNA processing and the Symp. Biol. 26, 240-266.
Ryskov, A. P., Saunders, G. F., Farashyan, V. R. and Georgiev, G. P. (1973). Double-helical regions in nuclear precursor of mRNA (pre-mRNA). Biochim. Biophys. Acta 312, 152-164. Ryskov, A. P., Kramerov, D.A., Limborskaya, S. A. and Georgiev, G. P. (1975). The structure of nuclear pre-mRNA: VII. Hybridization and renaturation properties of double-helical regions of premRNA. Molekularnaya Biologiya 9, 6-18. Schmid. C. W.. Manning, J. E. and Davidson, repeat sequences in the Drosophila genome.
N. (1975). Inverted Cell 5, 159-172.
Derman, E. and Darnell, J. E. (1974). Relationship of chain transcription to poly(A) addition and processing of hnRNA in HeLa cells. Cell 3, 255-264. Fedoroff, N. V. and Wall, T. R. (1976). Complementary sequences in heterogeneous nuclear RNA. In Molecular Mechanisms in the Control of Gene Expression,d, W. J. Rudder, D. P. Nierlich and C. F. Fox, eds. (New York: Academic Press), p. 379.
Spohr. G.. Mirault, M. E., Imaizumi. T. and Scherrer. K. (1976). Molecular weight determination of animal cell RNA by electrophoresis in formamide under fully denaturing conditions on exponential polyacrylamide gels. Eur. J. Biochem. 62, 313-322.
Franklin, R. M. (1966). Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc. Nat. Acad. Sci USA 55, 1504-l 511.
Weissmann. C., Feix, G. and Slor, H. (1968). phage RNA: the nature of the intermediates. Symp. Quant. Biol. 33, 83-100.
Georgiev, G. P.. Ryskov, A. P., Coutelle, C., Mantieva, V. L. and Avakyan, E. R. (1972). On the structure of transcriptional unit in mammalian cells. Biochim. Biophys. Acta 259, 259-283. Hagenbuchle, O., Schibler, U. and Wyler, T. (1975). Intermolecular renaturation of hairpin loops leads to stable and specific aggregates of 26s rRNA and precursor rRNAs from Xenopus laevis. Eur. J. Biochem. 60, 73-82. 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. Biol. 18, 499-515. Helmkamp, G. K. and Ts’o, P. (1961). The secondary nucleic acids in organic solvents. J. Am. Chem. Sot. Holmes, D. S. and Bonner, J. (1973). Preparation, weight, base composition, and secondary structure clear ribonucleic acid. Biochemistry 72, 2330-2338.
structures of 83, 138-142. molecular of giant nu-
Imaizumi, T., Diggelmann, H. and Scherrer, K. (1973). Demonstration of globin messenger sequences in giant nuclear precursors of messenger RNA of avian erythroblasts. Proc. Nat. Acad. Sci. USA 70, 1122-l 126. Jelinek, W. and Darnell, heterogeneous nuclear Sci. USA 69, 2537-2541.
J. E. (1972). Double-stranded RNA from HeLa cells. Proc.
regions in Nat. Acad.
Kohne, D.. Levison, S. and Byers, M. (1977). A room temperature method for increasing the rate of DNA reassociation by many thousand-fold: the phenol emulsion reassociation technique (PERT). Biochemistry, in press. McKnight, G. S. and Schimke, R. T. (1974). Ovalbumin messenger RNA: evidence that the initial product of transcription is the same size as polysomal ovalbumin messenger. Proc. Nat. Acad. Sci. USA 71, 4327-4331. Macnaughton. M., Freeman, K. B. and Bishop, J. 0. (1974). A precursor to hemoglobin mRNA in nuclei of immature duck red blood cells. Cell 1, 117-125. Mayo, V. S. and de Kloet, S. R. (1971). Disaggregation of “giant” heterogeneous nuclear RNA of mouse Ehrlich ascites cells by thermal denaturation in the presence of formaldehyde. Biochim. Biophys. Acta 247, 74-79. Morrison, W. and Busch, H. (1973). Studies on sedimentation characteristics of heterogeneous nuclear RNA. Physiol. Chem. Phys. 5, 403-422. Nikolaev. N.. Schlessinger. D. and Wellauer. P. K. (1974). 305 preribosomal RNA of Escherichia coli and products of cleavage by ribonuclease Ill: length and molecular weight. J. Mol. Biol. 86. 741-747.
Strauss, J. H., Kelly, R. B. and Sinsheimer, R. L. (1968). Denaturation of RNA with dimethyl sulfoxide. Biopolymers 6, 793-807.
Wellauer, P. K. and Dawid. I. B. (1973). of RNA: processing of HeLa ribosomal USA 70, 2827-2831.
In vitro synthesis of Cold Spring Harbor
Secondary RNA. Proc.
structure maps Nat. Acad. Sci.
Wellauer. P. K. and Dawid, I. l3. (1974). Secondary structure of ribosomal RNA and DNA: I. Processing of Xenopus ribosomal RNA and structure of single-stranded ribosomal J. Mol. Biol. 89, 379-395. Zimmerman, S. B. and Sandeen. activity of crystallized pancreatic them. 74. 269-277.
G. (1966). The deoxyribonuclease.
maps laevis DNA.
ribonuclease Anal. Bio-
10, 597-610,
April
1977,
Copyright
@ 1977 by MIT
Intermolecular Duplexes RNA from HeLa Cells Nina Fedoroff Carnegie Institution of Washington Department of Embryology 115 West University Parkway Baltimore, Maryland 21210 Peter K. Wellauer Swiss Institute for Experimental Cancer Ch. des Boveresses CH-1066 EPALINGES s/Lausanne Switzerland Randolph Wall Molecular Biology Institute University of California Los Angeles, California 90024
Research
Summary Rapidly sedimenting hnRNA complexes contain regions of stable intermolecular duplex. Disruption of such complexes, as judged by a reduction in sedimentation rate, requires conditions sufficient to denature the duplex regions. Rapidly sedimenting molecules reappear only when the complementary sequences reanneal-that is, the formation of such complexes is dependent upon time and the concentration of homologous RNA. These experiments lead us to the conclusion that rapidly sedimenting hnRNA complexes consist of two or more largely single-stranded RNA molecules held together by short duplex regions. Precisely such structures have been visualized in the electron microscope. Rapidly sedimenting fractions of native nuclear RNA from preparative sucrose gradients consist primarily of large, multimolecular complexes interconnected by duplex regions averaging 300 base pairs in length. Exposure of the RNA to severely denaturing conditions eliminates such complexes. Reannealing of the RNA reconstitutes complexes which are indistinguishable from those observed in preparations before denaturation. Introduction It has often been noted that exposure of eucaryotic heterogeneous nuclear RNA (hnRNA) to denaturing conditions effects a moderate to extreme reduction in its size, as judged by sedimentation rate or mobility on polyacrylamide gels (de Kloet, Mayo and Andrean, 1970; Mayo and de Kloet, 1971; Imaizumi, Diggelmann and Scherrer, 1973; Morrison and Busch, 1973; McKnight and Schimke, 1974; Macnaughton, Freeman and Bishop, 1974; Derman and Darnell, 1974; Spohr et al., 1976). Large, denaturable hnRNA complexes have been viewed variously
in Heterogeneous
Nuclear
as aggregates of unrelated molecules and as “nicked” or “processed” primary transcripts (de Kloet et al., 1970; Mayo and de Kloet, 1971; Derman and Darnell, 1974). Scant attention has been paid to defining the physical basis of hnRNA complex formation, a necessary prerequisite to understanding the origin and significance of such complexes. In this communication, we report experiments designed to test the hypothesis that hnRNA complexes are partial duplexes. It is apparent from several studies that hnRNA complexes are quite stable, readily surviving under conditions sufficient to dissociate the kinds of aggregates observed to form between bacterial RNAs (Hayes, Hayes and Guerin, 1966; Mayo and de Kloet, 1971; Morrison and Busch, 1973; Spohr et al., 1976). We have already reported that HeLa hnRNA, as it is extracted from the cell, contains intermolecular duplexes, as well as complementary sequences not in double-stranded form (Fedoroff and Wall, 1976). Here we establish a relationship between the sedimentation behavior of hnRNA and its content of intermolecular duplexes. We conclude that rapidly sedimenting hnRNA molecules are primarily multimers interconnected by short, rather stable, double-stranded regions. Such structures have been visualized in the electron microscope and predominate in rapidly sedimenting fractions of hnRNA. Results Evidence That Denaturable hnRNA Complexes Are Intermolecular Partial Duplexes We isolated HeLa hnRNA by two different methods. Method 1 is a modification of the procedure developed by Holmes and Bonner (1973), and involves lysis of nuclei in a strongly denaturing medium prior to phenol extraction of the nucleic acids (method 1). We have also employed the more widely used procedure developed by Penman (1966), in which the chromatin is disrupted with DNAase before phenol extraction (method 2). HeLa hnRNA extracted by method 1 contains a much higher fraction of rapidly sedimenting material than hnRNA extracted by method 2. This is apparent from the data given in Table 1. Before denaturation, more than half the hnRNA sample prepared by method 1 had an apparent sedimentation coefficient >65, and almost a quarter of it had an apparent sedimentation coefficient >lOO. The fraction of the RNA sedimenting with or ahead of 45s prerRNA varied between 60 and 90% in different preparations isolated by method 1. Less than 15% of the undenatured hnRNA sample prepared by method 2 sedimented between 65s and lOOS, and almost none had an apparent S value in excess of 100. RNA sedimenting ahead of 455 pre-rRNA has a
Cell 598
Table
1. Duplex
Content
of hnRNA
Differing
in Sedimentation
Properties % Duplex’
RNA Extraction Method
Method
1
2
Procedure
Sedimentation
Range”
% Total
MateriaP
Native
Denatured
65S-100s
32
5.0
1.4
>I00
23
5.7
1.7
32S-45s
37
1.4
1.2
5OS-65s
9
4.6
1.6
13
4.9
1.4
65S-100s a Calculated with respect to rRNA standards. b Fraction of total labeled material in the indicated S value range. r Determined by RNAasedigestion without (native) or with (denatured) The duplex content was assayed by cellulose-ethanol chromatography. hnRNA was extracted from HeLa cells labeled for 2 hr with JH-uridine
higher duplex content than RNA sedimenting more slowly than 45s pre-rRNA (Table 1). Preliminary denaturation of RNA prepared by either method yields material sedimenting predominantly between the 28s and 45s rRNA markers on nondenaturing gradients (see Figure 2 below for an example). Preliminary denaturation also reduces the duplex content to ~2% (Table 1). These results suggest that double-stranded regions stabilize large complexes. Rapidly sedimenting hnRNA complexes dissociate concomitantly with melting of the duplex regions. The concentration of dimethyl sulfoxide (MeSO) required to denature RNA at room temperature is a function of its melting temperature (Strauss, Kelly and Sinsheimer, 1968). Samples of rapidly sedimenting hnRNA were therefore exposed to increasing concentrations of MeSO at 25°C. Parallel aliquots were withdrawn for analysis of sedimentation behavior and assay of duplex content. The results of this experiment are shown in Figure 1. The sedimentation data are represented, for convenience, by the fraction of material sedimenting with or ahead of 45s pre-rRNA. There is a concomitant decrease in duplex content and sedimentation rate between 60 and 80% MeSO. This is in the range expected for double-stranded RNA and higher than necessary to denature either single-stranded RNA or double-stranded DNA (Helmkamp and Ts’o, 1961; Strauss et al., 1968). Rapidly sedimenting hnRNA complexes reform under conditions favoring renaturation of intermolecular duplexes. Highly labeled hnRNA was mixed either with excess unlabeled hnRNA or unlabeled E. coli RNA and denatured with MeSO. Each sample was concentrated by ethanol precipitation, resuspended in a buffer of high ionic strength and annealed at 55°C. Aliquots were withdrawn at 2, 7.5 and 20 min for analysis on nondenaturing sucrose gradients. The results are shown in Figure 2. There is a marked shift in the sedimentation profile with time in samples containing a high hnRNA concen-
preliminary
heating
and fractionated
in 0.01 M Tris (pH 7.4). 0.001 M EDTA at 100°C. on nondenaturing
sucrose
gradients.
80
I 60 2 u rn : Al 4o
I
I
20 40 % OIMETHY
I
L
$
1
60 80 SULF OXIDE
Figure 1. Effect of Increasing Dimethyl on the Sedimentation Rate and Duplex
Sulfoxide Concentration Content of hnRNA
Nuclear RNA was purified by method 1 from HeLa cells labeled for 2 hr with JH-uridine in the presence of 0.04 pg/ml actinomycin D, then layered on a nondenaturing 15-30% sucrose gradient and centrifuged in an SW56 rotor at 56,000 rpm for 75 min at 25°C. Material which pelleted was dissolved in 0.01 M Tris (pH 7.4). 0.001 M EDTA. ethanol-precipitated and redissolved in 0.1 M NaCI. 0.05 M Tris (pH 6.85), 0.001 M EDTA. Aliquots were diluted IO fold into ice-cold aqueous Me&O to give the indicated final concentrations and incubated at 25°C for 10 min. Aliquots were withdrawn for determination of duplex content by celluloseethanol chromatography after RNAse digestion (see Experimental Procedures) and for sedimentation on nondenaturing sucrose gradients. The results are expressed as the fraction of material sedimenting with or ahead of the 32P-45S pre-rRNA marker included in each gradient (A-A) and as the fraction of total radioactivity in a ribonuclease digest eluting at the position of duplex RNA from cellulose (O---O).
Duplexes 599
in hnRNA
tration. By 2 min of incubation, the fraction of material sedimenting with or ahead of 4% prerRNA has almost doubled. By 20 min, almost 80% of the RNA sediments with or ahead of 45s prerRNA. No such shift was observed in control samples containing unlabeled E. coli RNA. After 20 min of incubation, there is no detectable shift in the sedimentation profile (Figure 2D, broken line). (The total RNA concentration was lower by a factor of 5 in the control containing E. coli RNA. Hence the terminal time point represents a 4 min incubation at an RNA concentration equivalent to that in the experimental sample.) Since the annealing times were the same in both experimental and control samples, the shift in sedimentation coefficient in the former cannot be attributed to intramolecular renaturation (Schmid, Manning and Davidson, 1975). The shift in sedimentation profile requires sequences present in hnRNA, but not in E. coli RNA, ruling out nonspecific aggregate formation. Since about half the sequences that can anneal in hnRNA would have annealed after incubation for 20 min at the high hnRNA concentration (Fedoroff and Wall, 1976), it appears probable that the formation of rapidly sedimenting complexes is consequent on annealing of complementary sequences. Since only a small fraction of the hnRNA becomes double-stranded, while the sedimentation profile of virtually the entire population is affected, it follows that the complexes consist of largely singlestranded molecules held together by short duplex regions. There is rapid formation of complexes sedimenting much faster than would be expected for dimers. This suggests that a given hnRNA molecule contains more than one short sequence capable of finding a complementary sequence on another molecule. Some Properties Thermal melting Figure 2. The Sedimentation
FRACTION
of the Duplex Regions in hnRNA curves for the intermolecular du-
Effect of Reannealing Properties
Denatured
hnRNA
on
Its
Nuclear RNA was prepared as described in the legend to Figure 1, except that all the material sedimenting with or ahead of 45s prerRNA was pooled during the initial gradient purification. Aliquots of labeled hnRNA (5 x 10’ cpm. 1.6 x lo6 cpmlpg) were mixed with 20 pg of unlabeled hnRNA in 0.01 M Tris (pH 7.4), 0.001 M EDTA, adjusted to 75% Me&O and heated at 55°C for 3 min. The RNA was then ethanol-precipitated, dissolved in HzO, adjusted to 0.5 mg/ml RNA in 0.4 M NaCI, 0.2 M Tris (pH 6.85),0.004 M EDTA. 10% formamide, and incubated for (A) 0 min, (6) 2 min. (C) 7.5 min and (D) 20 min at 55°C (04). Controls were treated identically, except that unlabeled hnRNA was replaced by unlabeled E. coli rRNA. and the annealing reaction was carried out at a final RNA concentration of 0.1 mg/ml. Only the 20 min time point is shown (D) (0- - -0). The samples were layered on nondenaturing lo30% sucrose gradients and centrifuged in an SW41 rotor at 40,000 rpm for 130 min at 25°C. 32P-45S pre-rRNA was included in each gradient as an internal marker. Sedimentation is from right to left.
Cell 600
plexes are shown in Figure 3. The T, values obtained were 77°C in 0.1 x SSC and 95°C in 1 x SSC. To follow the dissociation only of intermolecular duplexes, undenatured hnRNA was heated to the indicated temperatures, chilled and RNAase-digested. Double-stranded material was then assayed by cellulose-ethanol chromatography (see Experimental Procedures). This permits intramolecular duplexes to reform and excludes them from the measurement. Of the initial duplex population, 25-30% remains double-stranded in such an experiment. Brief incubation at the low hnRNA concentration used did not further increase this value (data not shown). The melting curves are quite sharp, and the T, values are virtually identical to those obtained with double-stranded RNA prepared by self-annealing in vitro transcripts of denatured bacteriophage T4 DNA. This duplex RNA gave T, values of 79°C in 0.1 x SSC and 95°C in 1 x SSC. A similar duplex RNA standard prepared using adenovirus type 2 DNA as template gave T,,, values of 86°C and 101°C at the two salt concentrations, respectively. The difference in the melting temperatures of the RNA standards reflects the difference in the GC content of the DNA templates used (Billeter, Weissmann and Warner, 1966). The GC content of T4 DNA is 34% and that of adenovirus DNA is 55%. Since the melting curves for the intermolecular duplexes in hnRNA are relatively
-I B
I
I
70
90
TEMPERATURE Figure 3. Thermal hnRNA
Melting
Curves
,
I
70
90
(“C)
of Intermolecular
Duplexes
in
Nuclear RNA was prepared as described in the legend to Figure 1, but not fractionated. Total, undenatured hnRNA was heated in 0.1 x SSC (A) or 1 x SSC (B), diluted with 0.2 M NaCI. 0.1 M Tris (pH 6.65), 0.002 M EDTA, RNAase-digested and subjected to cellulose-ethanol chromatography. The preparation used in this experiment contained 5% double-stranded RNA (100%). Intact hnRNA was used in this experiment to permit reformation of intramolecular duplexes before RNAase digestion, excluding them from the measurement. Roughly one quarter of the total double-stranded RNA behaved as intramolecular duplexes.
sharp, the low T, probably reflects a low GC content for the duplex population. The sequences which are double-stranded in undenatured hnRNA appear to be the most abundant complementary sequences in hnRNA. Doublestranded RNA was isolated by cellulose-ethanol chromatography from undenatured hnRNA prepared by method 1 and digested with RNAase. The recovered double-stranded material was denatured and reannealed to a Rot of 0.15 M.sec (normalized to the initial hnRNA concentration). The sample was then separated into double-stranded and single-stranded fractions by cellulose-ethanol chromatography without RNAase digestion. The fraction remaining single-stranded was further annealed to a Rot of 15 M set and refractionated. Of the total complementary sequences in hnRNA, roughly 17% are double-stranded at a Rot of 0.15 M.sec (Fedoroff and Wall, 1976), as compared with 38% for the renatured duplexes. At the higher Rot of 15 M.sec, roughly half the total complementary sequence population is double-stranded (Fedoroff and Wall, 1976), while 78% of isolated duplexes have reannealed. Although these experiments are not completely comparable, since isolated, reannealed double-stranded RNA was fractionated without RNAase treatment, they nonetheless suggest that complementary sequences which are double-stranded in hnRNA as it is isolated from the cell are those that are most abundant in the total complementary sequence population. The complementary sequences that are doublestranded in undenatured hnRNA are almost exclusively transcripts of highly reiterated DNA. This was determined by hybridizing the Rot-fractionated duplexes obtained in the preceding experiment to a 300,000 fold excess of human placental DNA. The three fractions whose hybridization curves are shown in Figure 4 represent material reannealing at values of Rot up to 0.15 M.sec, at values of Rot between 0.15 M.sec and 15 M.sec, and at values of Rot >15 M.sec. Hybridization was assayed by the development of RNAase resistance. Reassociation of the RNA was minimized by the choice of conditions and monitored by the inclusion of controls lacking DNA, but containing RNA. The RNAase resistance developed in control samples is plotted in the lower curves of Figure 4 as a function of the Cot attained in DNA-containing experimental samples incubated for the same length of time. The upper curves show the RNAase resistance in experimental samples as a function of Cot. About 90% of the observed hybridization occurs at Cot values below 10 Msec. Only the most slowly annealing fraction contains material which hybridizes at higher Cot values (Figure 4C). Hence the complementary sequences that are duplexed in native hnRNA are
Duplexes 601
in hnRNA
10-1
10-Z
10-l
1
10
102
IO'
CO'
Figure 4. Hybridization to DNA of Rot-Fractionated Derived from Undenatured hnRNA
Duplex
RNA
Nuclear RNA, labeled as described in the legend to Figure 1, and sedimenting with or ahead of 455 RNA in a preparative, nondenaturing sucrose gradient, was digested with RNAase. The duplex RNA was purified by cellulose-ethanol chromatography. The double-stranded RNA was heat-denatured and reannealed to a Rot of 0.15 M’sec (based on the initial hnRNA concentration), then separated into single- and double-stranded fractions by celluloseethanol chromatography without RNAase digestion. The singlestranded fraction was further annealed to a Rot of 15 M.sec, and the fractionation was repeated. Aliquots were then hybridized to a 300,000 fold excess of sheared human placental DNA in phosphate buffer at 60°C and assayed for ribonuclease resistance developed in the presence (upper curves) or absence (lower curves) of DNA. Control values of ribonuclease resistance are plotted as a function of the Cot attained in experimental tubes incubated for the same length of time. (A) hnRNA reannealing at Rot%O.lS M.sec; (8) hnRNA reannealing at 0.15sRotG15 M.sec; (C) hnRNA reannealing at Rot>15 M’sec.
transcribed from highly reiterated DNA. This is, once again, by contrast with the total complementary sequence population in hnRNA, which contains transcripts of DNA sequences present at high, moderate and low reiteration frequences (Fedoroff and Wall, 1976). Electron Microscopic Analysis of hnRNA The basic difficulties in analyzing hnRNA by electron microscopy proved to be the very large size of the hnRNA complexes and the varied array of structures. hnRNA spread from 30% formamide/ M urea showed extensive secondary structure, as well
as intermolecular associations. Many molecules were almost completely collapsed into loops and hairpins. Increasing concentrations of formamide/ urea gradually eliminated secondary structure. Molecules apread from 90% formamide/ M urea had little secondary structure, yet most molecules remained in multimolecular complexes. The hnRNA preparation used here was purified by method 2. The RNA sedimenting ahead of 45s pre-rRNA after a single round of centrifugation was found to contain structures characteristic of rRNA (Wellauer and Dawid, 1973). It was therefore repurified on a second sucrose gradient, and molecules having an apparent S value range of 65-100 were pooled and used for electron microscopy. Electron micrographs of undenatured hnRNA molecules are shown in Figures 5 and 6. RNA was spread from 90% formamide/4.5 M urea. The complexes shown contain three or four distinct singlestranded RNA molecules connected by short duplex regions. Each complex contains at least double-stranded regions. Although the duplex regions are short and strikingly uniform in length, fairly long ones are occasionally observed (Figure 5). In measuring duplex lengths, we have omitted from consideration crossover points such as those indicated by broken arrows in Figure 5, since these cannot reliably be identified as duplexes. The single-strand distance separating two duplex regions is highly variable, as are the lengths of the single-stranded tails emerging from a duplex region. The molecules shown here are particularly well spread out. Even when spread from 90% formamide/4.5 M urea, most complexes are sufficiently tangled to preclude enumeration of the molecules with certainty. Because of this, we cannot make meaningful quantitative statements either about the frequency of such complexes or about the number of molecules per complex. Qualitatively, however, we can state that most of the hnRNA mass is in such complexes when the RNA is spread from 90% formamide/4.5 M urea. Preliminary denaturation eliminates large complexes. Occasional dimers remain. Such structures were not included in length measurements. A histogram showing the lengths of denatured hnRNA molecules is shown in Figure 7. The calculated number average length is about 4100 nucleotides, and the weight average length is 7100 nucleotides. An aliquot of the denatured preparation used to generate the length histogram in Figure 7 was renatured for 10 min at 55°C in 0.4 M NaCl, 0.2 M Tris (pH 6.85), 0.004 M EDTA, 10% formamide and at a concentration of about 150 pg/ml before spreading. It is essentially indistinguishable from the native preparation. A relatively well spread complex from the renatured preparation is shown in Figure
Cell 602
Figure
5. Electron
Micrograph
of an hnRNA
Complex
Spread
from
90% Formamide/4.5
M Urea
HeLa hnRNA was extracted by method 2 from cells treated with actinomycin D to suppress rRNA synthesis. Material sedimenting ahead of 45s rRNA on a nondenaturing sucrose gradient was pooled and rerun on a second sucrose gradient. Material running in the 65S-100s region was pooled, ethanol-precipitated twice and then redissolved in 10 mM Tris (pH 7.5) for spreading. The tracing (b) shows our interpretation of the photograph (a). Single-stranded RNA is drawn as a thin line and double-stranded RNA as a thick line. Single-stranded ends are indicated by solid arrows and intermolecular duplexes by open arrows. An additional region which may comprise a short duplex is indicated by the broken arrow. Such structures were observed frequently, but were omitted from measurements of duplex length (Table 2), since they are not distinguishable in the electron micrographs from fortuitous crossing-over of free RNA molecules. Bar = 0.1 pm.
Duplexes 603
in hnRNA
b Figure
6. Electron
The details
Micrograph
of hnRNA
preparation
of an hnRNA
Molecule
are given
in Figure
Spread
from
90%
FormamidW4.5
M Urea
5. Bar = Cl.3 pm.
8. It should be noted that renaturation reconstitutes complexes containing more than two molecules, as expected from the results of the experiment represented in Figure 2. In Figure 9, we show the different kinds of duplex structures encountered in native hnRNA. Since the preparations were spread from 90% formamide/
urea, these represent the most stable inter- and intramolecular duplexes in the RNA. These structures include hairpins and loops (a and b), forks and duplexes with four single-stranded tails of various lengths (c and d). The duplex lengths were measured for two broad categories, intra- and intermolecular duplexes. Included in the intramolec-
Cell 604
LENGTH
Figure Cells
7. Length
Distribution
(pm)
Histogram
of hnRNA
from
HeLa
The RNA was denatured by boiling in 10 mM Tris (pi-i 7.5) for 2 min and spread immediately from 90% formamide/4.5 M urea. The number average length of 630 molecules is 1.29 pm, or about 4100 nucleotides. The weight average length is 2.24 pm. corresponding to about 7100 nucleotides. The length was determined by comparison with E. coli 23s rRNA. which was spread separately under similar conditions. The few small complexes that were still present after denaturation were omitted from these measurements.
ular category were all loops and hairpins. Other structures were considered intermolecular. This distinction between intra- and intermolecular is somewhat arbitrary in the case of hairpins, since discontinuities in primary structure within such regions cannot be distinguished in the electron microscope. The lengths of intra- and intermolecular duplexes, respectively, in native hnRNA spread from 90% formamide/ urea are given in Table 2. Both have an average length of about 0.1 p, which corresponds to about 300 base pairs. The lengths of intermolecular duplexes in native hnRNA spread from 30% formamide/l.5 M urea, and in denatured and reannealed hnRNA spread from 90% formamide/4.5 M urea are also given in Table 2. The average length is the same in all cases. The length of double-stranded RNA isolated after ribonuclease digestion was determined by polyacrylamide gel electrophoresis to be in the range of 50-200 base pairs (data not shown). Assuming that the RNAaseresistant duplexes are representative of those observed by electron microscopy, this result indicates the presence of RNAase-sensitive mismatches within the double-stranded regions. Discussion We have been quite impressed with the difficulty of obtaining hnRNA completely free of partially duplexed structures. hnRNA samples exposed to 90% MeSO or heated to 100°C in 10 mM Tris (pH 7.5), then ethanol precipitated before spreading for electron microscopy, contained a few dimers. Surprisingly, even an hnRNA sample boiled in water
immediately before spreading contained dimers. Such residual partial duplexes might represent regions which have not denatured, either because of their base composition or because they are crosslinked in some way. Alternatively, and perhaps more probably, such duplexes might result from the renaturation of some of the complementary sequences on different RNA molecules. Reannealing of complementary sequences is observed at Rot values between 10e3 and lO-2 M.sec (Fedoroff and Wall, 1976). Significant reassociation at room temperature might easily be expected within a period of a few minutes at the formamide and urea concentrations used during RNA spreading for electron microscopy. We believe that the complementary sequences studied here are not generally double-stranded in the cell. This contention rests on the observation that hnRNA prepared by two different methods from the same cells differs markedly in duplex content. Correlated with the difference in duplex content is a marked difference in sedimentation behavior-that is, the higher the content of intermolecular duplexes, the more rapidly does the RNA sediment in a sucrose gradient. Pronounced variation in the sedimentation profiles of undenatured hnRNA is the rule rather than the exception in the published literature (Bramwell and Harris 1967; Georgiev et al., 1972; Morrison and Busch, 1973; Derman and Darnell, 1974; Macnaughton et al., 1974). Perhaps the basis of this variation is the extent to which complementary sequences normally not duplexed in the cell are able to anneal during isolation and manipulation of the hnRNA. Supporting evidence that similar sequences can be either single- or double-stranded in hnRNA as it is isolated comes from the following observations. Ryskov et al. (1975) reported that when labeled, double-stranded RNA isolated from murine ascites hnRNA was denatured and reannealed, its renaturation rate could be accelerated by the addition of unlabeled and undenatured hnRNA. This means that undenatured hnRNA contains, in singlestranded configuration, sequences which can also be present in double-stranded form. There are several large RNAase Tl oligonucleotides highly characteristic of duplex regions in HeLa hnRNA (H. D. Robertson, W. Jelinek and E. Dickson, personal communication). A smaller fraction of these sequences is cleaved by the double-strand-specific RNAase III in total hnRNA than in isolated hnRNA duplexes (H. D. Robertson et al., personal communication). In addition, hnRNA recovered from DNARNA hybrids obtained at low values of Cot gives the Tl oligonucleotide pattern characteristic of double-stranded regions. However, much more of the hnRNA is recovered from such hybrids than is present in the form of double-stranded RNA (R. Evans,
Duplexes 605
in hnRNA
cI 7
-
-? I
I b
Figure
6. Electron
Micrograph
of a Complex
in a Denatured
and Reannealed
hnRNA
Sample
Spread
A sample of hnRNA, prepared as described in Figure 5, was denatured by heating to 100°C in 10 mM was adjusted to 0.4 M NaCI, 0.2 M Tris (pfi 6.65), 0.004 M EDTA, 10% formamide, and the sample was was precipitated with ethanol, redissolved in IO mM Tris (pfi 7.5) and spread from 90% formamide/4.5 at least six individual molecules joined by five duplex regions (open arrows). Single-stranded tails secondary structure is also seen in this particular complex. The insert shows a small dimeric molecule of hnRNA. Bar = 0.3 &rn.
from
90% Formamide/4.5
M Urea
Tris (pfi 7.5). The salt concentration annealed for 10 min at 55°C. The RNA M urea. The complex shown contains are indicated by solid arrows. Some from the same renatured preparation
Cell 606
Figure
9. Electron
Micrographs
and Tracings
of Various
Types
of Duplex
Structures
Observed
in hnRNA
(a) photographs of intramolecular duplexes. These are double-stranded structures, without or with a terminal single-stranded loop, and with two single-stranded tails. (c) photographs of intermolecular duplexes. These include, from left to right, forked structures, forked structures with one or two short whiskers and structures with four long single-stranded tails. In (e) and (f), intramolecular (double arrows) and intermolecular (single arrows) duplexes are shown for comparison in the same molecule. Note that the two types of duplexes are of similar length. The tracings (b and d) show our interpretation of the various structures. Single-stranded RNA is represented by thin lines and double-stranded RNA by hatched bars to indicate hydrogen bonding. Long single-stranded tails are indicated by dashes. The bar of 0.1 pm corresponds to about 300 bases/base pairs.
Duplexes 607
Table
in hnRNA
2. Size of Duplex
Regions
in HeLa
Cell hnRNA Spreading Conditions
L, + SD pm (Number Molecules)”
of
Number Pairs”
RNA
Type of duplex
Native
Intermolecular
30% formamide, 1.5 M urea
0.091
+ 0.036
(146)
-290
Native
Intermolecular
90% formamide. 4.5 M urea
0.097
+ 0.049
(112)
-310
Reannealed
Intermolecular
90% formamide. 4.5 M urea
0.090
+ 0.026
(156)
-290
Native
Intramolecular’
90% formamide, 4.5 M urea
0.083
f 0.032
(138)
-260
of Nucleotide
a L, = number average length. ’ E. coli 23s rRNA (3070 nucleotides; Nikolaev, Schlessinger and Wellauer, 1974) spread under similar conditions has an average linear density of 1 .l x lo6 daltons per pm. The single-strand molecular weight of duplexes in hnRNA was obtained by multiplying length with linear density of E. coli rRNA. Duplex and single-stranded RNA have very similar linear densities (Wellauer and Dawid, 1973) under the spreading conditions used. The number of nucleotide pairs was determined by dividing the molecular weight of duplexes by the average molecular weight per nucleotide. ’ In view of the spreading conditions used, the intramolecular duplexes measured here represent the most stable ones in hnRNA. The two types of secondary structure features that were measured are fully double-stranded structures and structures with a double-stranded stem and a loop at the end (see Figure 9); only the stem was measured in structures with loops.
personal communication). Once again, these observations suggest that not all of this simple complementary sequence population is doublestranded in the hnRNA as it is extracted from the cell. There is ample reason to believe that the environment to which the RNA is exposed during extraction promotes the annealing of complementary sequences. In particular, it has recently been shown that annealing reactions are markedly accelerated in a two-phase, phenol-aqueous system (Kohne, Levinson and Byers, 1977). There are two quite well documented examples of highly specific RNA annealing reactions which occur during extractions involving phenol. These are the stable partial duplexes formed by 28s rRNAs and pre-rRNAs of Xenopus laevis tissue culture cells (Hagenbuchle, Schibler and Wyler, 1975) and the double-stranded structures formed by complementary, replicating bacteriophage RNAs during isolation (Weissmann, Feix and Slor, 1968). But in both these instances, and probably in the case of hnRNA, where we have obtained preparations consistently differing in duplex content by two methods both involving phenol extraction, the explanation is undoubtedly more complex than merely phenol-mediated acceleration of annealing. Such parameters as local concentrations of complementary sequences at the time of deproteinization may well be important. Simple reconstruction experiments, where they have been tried, have failed (Weissmann et al., 1968; Hagenbuchle et al., 1975). It has been suggested that denaturable hnRNA complexes are ‘nicked” or “processed” primary transcripts (Derman and Darnell, 1974). The implication here is that complexes contain contiguous transcripts whose summed length approximates
that of the transcription unit. In the preceding discussion, we have argued that many, if not all, of the denaturable complexes are formed during isolation and manipulation of the RNA. This could be true, yet not preclude formation of highly specific complexes containing only contiguous transcripts. If such specific complexes did form during isolation of the RNA, their sequence composition would be very different from that of complexes formed during in vitro reannealing of denatured hnRNA. This follows from the fact that there are many copies of relatively few complementary sequences. H. D. Robertson et al. (personal communication) have estimated that the total complexity of the duplex regions in HeLa hnRNA is < 1000 nucleotides. By kinetic means, we have estimated that the simplest complementary sequence component in hnRNA has a complexity of roughly 1500 base pairs (Fedoroff and Wall, 1976). It is therefore far more probable that upon renaturation, a given sequence anneals to a complementary region on a different molecule from the one it was initially joined to, than that it finds its initial complement. Despite this randomization, our blunt tools find the native and renatured preparations indistinguishable. Even in the electron microscope, they appear to contain the same kinds of multimolecular networks held together by the same range of duplex structures of the same length. This means either that all complexes are random partial duplexes or that specific complexes cannot be distinguished structurally from random complexes. The relationship between the sequences we have studied here and the “hairpin” RNA sequences described by other investigators is not clear (Jelinek and Darnell, 1972; Ryskov et al., 1973). Unless care is taken to distinguish kinetically between intramo-
Cell 606
lecular and rapid intermolecular duplex formation, any given population of double-stranded RNA isolated from hnRNA would tend to contain both kinds of sequences. There is no inherent reason, however, that the two populations need have a different sequence content. Sequences in intramolecular duplexes could be transcribed from a subset of the same family of highly reiterated sequences in DNA as those in intermolecular duplexes. Some members of this family of sequences might occur as inverted repetitions in close juxtaposition giving rise to “hairpinned” transcripts. In summary, our experiments show that heterogeneous nuclear RNA readily forms networks of molecules interconnected by double-stranded regions averaging 300 base pairs in length. This property of hnRNA renders suspect length measurements made under any but the most rigorously denaturing conditions (Spohr et al., 1976) and may preclude resolution of questions about the length of transcription units from analyses of purified transcripts. The complementary sequences which are duplexed in undenatured hnRNA are quite abundant, have a low overall sequence complexity and form stable duplexes. We wish here to focus attention on their simplicity and ubiquity. These properties suffice to make such sequences good candidates for cleavage sites in the reductive “processing” that has been postulated to occur in the synthesisof eucaryotic mRNAs (Darnell, Jelinek and Malloy, 1973). We believe it important, however, to consider the possibility that these simple, abundant sequences find their significance at other levels of genetic organization and expression. They could, at one extreme, reflect the molecular architecture of the genome and have no function as transcripts. At the other extreme, they could have an autonomous function in nuclear RNA metabolism. Theoretical constructs postulating specific roles for transcripts of reiterated DNA sequences have appeared recently (Robertson and Dickson, 1974; Davidson, Klein and Britten, 1977). Experimental
Procedures
Cell Labeling and Isolation of Nuclei HeLa cells growing in Eagle’s medium were concentrated to 4 x lO”/ml and incubated with 0.04 pg/ml actinomycin D for 25-30 min before labeling. 3H-uridine (5 mCi/lO ml cells, 29.2 Ci/mmole) was added, and incubation continued for 2 hr (Derman and Darnell, 1974). Labeling was terminated by pouring cells over frozen, crushed, phosphatebuffered saline. Unlabeled hnRNA was extracted from cells incubated in the presence of 0.04 pg/ml actinomycin D for 3 hr. Nuclei were prepared from labeled cells as described by Penman (1966). Unlabeled cells were concentrated by centrifugation,
resuspended at 10B/ml in cold 0.32 M sucrose (Schwarz/Mann, RNAase-free), 3.0 mM MgCI,, 500 pglml heparin (Riker Laboratories), and lysed in a Kontes Cell Disruption Bomb. The lysate was adjusted to 500 pg/ml spermidine and centrifuged at 2000 x g for 5 min. Nuclei were then detergentwashed as described by Penman (1966). hnRNA Extraction-Method 1 Nuclei were taken up in 1 ml/l-2 x 10’ starting cells of cold nuclear lysis solution containing 7 M urea (Schwarz/Mann, Ultrapure), 0.35 M NaCI, 0.01 M Tris (pH 7.6), 0.001 M EDTA, 2% Sarkosyl (M Chemical Co.), and 5% phenol (Mallinckrodt, giltlabel). The resulting viscous solution was gently homogenized in a Dounce homogenizer using a loose-fitting pestle. 1 vol of a 3:l mixture of phenol and chloroform containing 5% isoamyl alcohol was added, and the solution was brought quickly to 3040°C with shaking. Phases were separated by low speed centrifugation at room temperature. The phenol phase was reextracted with 0.5 vol of the nuclear lysis mixture, and the pooled aqueous phases were reextracted with phenol mixture, followed by dialysis against cold 0.1 M Na acetate (pH 7.0) in diethyl pyrocarbonate-treated dialysis tubing. The solution was adjusted to 0.01 M MgCI,and 10 pg/ml iodoacetate-treated DNAase (Zimmerman and Sandeen, 1966), incubated at 37°C for 15 min and phenol-extracted as above, following addition of EDTA to 0.01 M and sodium dodecylsulfate to 0.5%. This procedure is a modification of that described by Holmes and Bonner (1973). Ethanolprecipitated RNA was collected by centrifugation and washed with 3.0 M Na acetate (pH 6.0) to remove residual DNA fragments (Macnaughton et al., 1974), then with 0.2 M Na acetate (pH 6.0), 75% ethanol. hnRNA Extraction-Method 2 Nuclei were digested with iodoacetate-treated DNAase, and RNA was extracted as described Penman (1966).
by
Sucrose Density Gradient Sedimentation Nondenaturing gradients were 15-30% sucrose in a buffer consisting of 0.1 M NaCI, 0.01 M Tris (pH 7.4), 0.001 M EDTA and 0.1% SDS. After denaturation in MeSO, samples were diluted at least 3 fold with 0.01 M Tris (pH 7.4), 0.001 M EDTA before layering on such a gradient. Annealing RNA was denatured by heating to 100°C for 3 min in 0.01 M Tris (pH 7.4), 0.001 M EDTA. Annealing was carried out in 0.4 M NaCI, 0.2 M Tris (pH 6.85), 0.004 M EDTA, 10% formamide in sealed capillaries at 55°C. The extent of annealing was monitored
Duplexes 609
in hnRNA
by cellulose-ethanol RNAase digestion.
chromatography
following
RNAase Digestion RNA was digested with 0.5 pg Tl RNAase (Calbiochem) and 1 unit of T2 RNAase (Calbiochem) per 20 pg of RNA in 0.2 M NaCI, 0.1 M Tris (pH 6.85), 0.002 M EDTA. The RNA concentration was maintained at 50 pg/ml by addition of E. coli rRNA when necessary. RNAase-resistant material was measured by applying the digest directly to a celluloseethanol column. DNA-RNA Hybridization Isolated double-stranded hnRNA was hybridized to human placental DNA (a gift from Dr. M. Shoyab), sonicated to an average length of 500 nucleotides, in Na phosphate buffer (pH 6.8). Hybridization was carried out in 0.03 or 0.2 M phosphate buffer, and equivalent Cot values were calculated as described by Britten, Graham and Neufeld (1974). Hybrids and controls lacking DNA were supplemented with 20 pg E. coli rRNA, and digested with 1 pg of Tl RNAase and 1 unit of T2 RNAase in 0.3 M NaCI, 0.03 M Na citrate for 30-60 min at 37°C. Samples were precipitated with 5% CCICOOH, and precipitates were collected on millipore filters for liquid scintillation spectrometry. Cellulose-Ethanol Chromatography Chromatography on cellulose (Sigmacell Type 38, Sigma) was performed as described by Franklin (1966). Columns were equilibrated with a buffer consisting of 35% absolute ethanol and 65% 0.1 M NaCI, 0.05 M Tris (pH 6.85), 0.001 M EDTA; samples were applied in the same buffer. The columns were washed with 35% ethanol buffer, then with the same buffer containing 15% ethanol. Doublestranded RNA was eluted with buffer alone (0% ethanol). Analytical columns were prepared in siliconized Pasteur pipets. RNAase digests in 0.2 M NaCI, 0.1 M Tris (pH 6.85), 0.002 M EDTA were applied to columns after dilution with 1 vol of 70% ethanol. Fractions were collected directly into vials, mixed with Triton X-100 scintillation fluid and counted. Mono- and oligonucleotides do not bind to the columns in the 35% ethanol sample application buffer. Single-stranded RNA elutes in 15% ethanol, and double-stranded RNA elutes in buffer alone. On a preparative scale, double-stranded RNA was isolated from hnRNA by RNAase digestion as described above, followed by passage through two successive cellulose columns (approximately 1 ml packed bed volume per pg double-stranded RNA). In Vitro Synthesis of RNA RNA complementary to T4 and adenovirus type 2 DNAs was synthesized in a reaction mixture con-
taining 0.04 M Tris (pH 7.9), 0.05 M KCI, 0.01 M MgCI,, 0.1 mM dithiothreitol, 0.4 mM ATP and GTP, 0.13 mM CTP and UTP, 0.01 mM 3H-CTP (Schwarz/ Mann, 25 CVmmole), 0.01 mM 3H-UTP (SchwartzMann, 18.6 Ci/mmole), 110 pg/ml heat-denatured T4 DNA (a gift from Dr. D. Kaplan) or 100 pglml heat-denatured adenovirus type 2 DNA, and 120 pglml E. coli RNA polymerase (400 units per mg; a gift from Dr. W. Mangel). At the end of 2 hr at 37”C, iodoacetate-treated DNAase was added to 180 pgl ml, and incubation at 37°C continued for 20 min. The reaction mixture was phenol-extracted, and the RNA was freed from nucleotides by celluloseethanol chromatography. Double-stranded RNA was prepared by self-annealing, RNAase digestion and cellulose-ethanol chromatography as described above. Electron Microscopy RNA was spread for electron microscopy as described in detail earlier (Wellauer and Dawid, 1973, 1974). Formamide/urea solutions of varying denaturing power were obtained by appropriate dilution of stock solution of 5 M urea in 99% formamide (Fisher certified, used without further purification). The concentration of the remaining components in the final spreading solution were: 0.5 pglml of RNA, 30 mM Tris (pH 8.5), -1 mM EDTA and 30 wg/ ml of cytochrome c. The hypophase was distilled water. Pictures were taken in a Hitachi HU-11 El electron microscope at a magnification of 14,000X. For each set of pictures, the magnification of the instrument was calibrated with a carbon replica of a diffraction grating (2160 lines per mm). Negatives were enlarged in a projector to a final magnification of 300,000X and measured with a graphics calculator (Numonics). Acknowledgments We gratefully acknowledge the excellent technical assistance of Kathleen Toth. This work was supported by grants from the NIH. the California Institute for Cancer Research, and the Swiss National Science Foundation. Some of this work was performed during the tenure of a postdoctoral fellowship from the Damon Runyan-Walter Winchell Fund for Cancer Research (N. F.). Received
October
12, 1976; revised
December
17, 1976
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Spohr. G.. Mirault, M. E., Imaizumi. T. and Scherrer. K. (1976). Molecular weight determination of animal cell RNA by electrophoresis in formamide under fully denaturing conditions on exponential polyacrylamide gels. Eur. J. Biochem. 62, 313-322.
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