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Cellular Skeletons and RNA Messages
Cellular Skeletons and RNA Messages RONALDHERMAN, GARYZIEVE, JEFFREYWILLIAMS, ROBERTLENKAND SHELDONPENMAN Department of Biology Massaclztisetts Institute of Technology Cambridge, Massachuretts
Progress in the study of eukaryotic, and especially metazoan, cell biology is quite apparent from the contributions at this conference. Nevertheless, the challenge remains to elucidate those properties of gene expression, presumably through RNA metabolism, that serve to make a metazoan animal the complex arrangement of biological materials that it is. In particular, morphogenesis involves a bewildering variety of cell growth, movement, changes in architecture and the development of special biochemical pathways. Although the impressive work on the RNA metabolism of higher organisms has established profound differences from the metabolism of prokaryotes, so far, with few exceptions, little relates our studies to the obvious problems of metazoan biology. In this report, we describe some of our first tentative efforts to relate RNA metabolism to the unique properties of a mctazoan cell. W e present suggestive evidence that the architecture of the metazoan cell, in this case mammalian, is intimately involved with RNA metabolism.
1. Cytoplasmic Skeleton We describe two preparations (“skeletons”) from HeLa cells, one cytoplasmic and one nuclear. [The term “skeleton” is currently used to suggest a number of different cellular structures; perhaps in the near future a more definitive terminology will be adopted.] The choice of HeLa cells for a study of cell architecture may seem odd since these cells have little in the way of morphologically distinct features. Nevertheless, for other reasons, this work was started using this rather nondescript workhorse of a cultured cell, and a quite remarkable degree of internal structure was found. 379
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Figure 1 is a low-power clectron micrograph of a HeLa cell gently lysed with Triton X-100 in an isotonic buffer. The general structure of a cell and nucleus are clearly visible, and even such specialized morphological entities as surface microspikes seem to be partially preserved. What is remarkable about this preparation is how few of the cellular constituents remain. The procedure extracts most of the phospholipids and a11 of the soluble components, such as proteins and transfer RNA, and mitochondria1 constituents have apparently been leached out. Furthermore, the cold extraction procedure breaks down microtubules, and tubulin is quantitatively removed. Actin filaments are not visible; actin is found largely in extracted proteins either because of its unpolymerized state in these cells or owing to an instability of nonmuscle f-actin under these extraction conditions. What remains is comprised of a network of filaments that at higher magnification are seen to be the intcrincdiate filaments previously described ( 1-3). These apparently interconnect and possibly mesh with the as yet undefined proteins, which appear condensed in rather diffuse blotches. Thus, a major component of the structure that maintains morphology in the absence of microtubules or microfilaments appears to be the intermediate filaments. The analysis of “skeleton” proteins shows that the 53,000-dalton subunit of these filaments described by Shelanski and co-workers ( 4 ) is, in fact, a major component and is quantitatively rctained in the preparation. Most s t a r t h g is the retention of most of the active polyribosomes of the cell. These are quite apparent in the electron micrograph, and biochemical measurements show that at least 75% of the active cellular polyribosomes remain attached by some linkage to the cytoskeleton. In contrast to the active polyribosomes, the inactive monomers are largely extracted by the lysis procedure. The polyribosomes always appear to be associated with the blotches of condensed protein apparent in the cellular network. Where the intermediate fibers are particularly dense, the polyribosomes appear excluded. The most difficult thing to establish at this point is whether the association of polyribosomes with the cytoskeleton represents a truc in viuo state or is some artifact of the extraction procedure. The major evidence that this association may represent the true distribution of polyribosomes in the intact cell is the observation that ribosomal monomers do not stick to the “skeleton” to any significant degree. Of course, the active ribosomes may very well have components that lead to an artifactual association; at present, this possibility cannot be ruled out. Experiments are in progress to determine whether the products of the extracted polyribosomes are in any way differentfrom those that remain attached to the skeleton.
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FIG. 1. Cytaskeleton of HeLa cells. HeLa cells in suspension culture were harvested, washed and resuspended in isotonic, low ionic strength buffer (0.25 M sucrose, 0.01 M NaCI, 0.003 M MgCI,, 0.01 M Tris, pH 7.4) containing 1%Triton X-100. The cells were vortexed briefly and centrifuged into a pellet. Fixation was with 2.5% glutaraldehyde followed by 1%osmium tetroxide. The sample was dehydrated in alcohol, embedded in Epon-Araldite and sectioned. Micrographs were taken with a JEM lOOB at 80 Kev. The microscopy was carried out by Elaine Lenk.
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Taken at face value, and assuming that the preparation is not plagued by artifacts, the electron-microscope and biochemical studies suggest strongly that the protein synthetic machineiy of the cell does not float about freely but, even in the case of “free” (as opposed to membranebound) polyribosomes, is localized within the cytoplasm. This would serve to explain such puzzling morphological observations as the apparent exclusion of ribosomes from certain regions of the cell, such as the vicinity of the centriole, Also, the localization of the protein synthetic machinery on relatively spatially stable structures makes some teleological sense. In such cases as cell division or extensive ccll movement, a randomly diffusing protein synthetic system would be out of cellular control and the partition of polyribosomes between daughter cells or parts of extended cell would be left to chance. Also, there may be situations similar to those involving products of membrane-bound polyribosomes, where the spatial location of polyribosome products is important. All this is speculative and requires considerable further effort to demonstrate the reality of the suggested topological control of the proteinsynthesizing components of the mammalian cell.
II. The Nuclear Skeleton and hnRNA We return to the cytoskeleton below, but consider here another “skeleton”, this time the one associated with the nucleus and presumably related to the structures previously described by Berezney and Coffey ( 5). Figure 2 shows an early preparation in which about SM of the DNA has been removed by microccocal nuclease digestion. Much of the remaining DNA appears localized in the perinuclear heterochromatin, which appears to be relatively less accessible to nuclease. Later preparatory techniques, for which electron micrographs are not presently available, remove the remaining plasma membrane components surrounding the nuclear shell and more than 95% of the total nuclear DNA. Even in this early electron micrograph (Fig. 2 ) , it is possible to see that the nucleus retains its shape (somewhat distorted in this preparation by high centrifugation forces) and that suggestions of internal structure are visible through the empty space left by the removed chromatin. A prominent nucleolus is visible; it is possible that it is simply trapped by the nuclcar shell. We postulate a relation between nuclear metabolism, chromatin organization, and this nuclear skclcton. This interrelation is apparently accomplished by a class of large hnRNA molecules that are relatively long-lived, many terminating with poly( A ) , and that appear to be attached to both the skeleton and the chromatin.
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FIG.2 . Nuclear skeleton of HeLa Cells. HeLa cells were broken in hypotonic buffer (0.01 M NaCl, 0.003 M MgCl,, 0.01 M Tris, pH 7.4) to which 1%Triton X-100 was added. Nuclei were separated by centrifugation and resuspended in digestion buffer ( 5 % sucrose, 10.' CaCl?, 0.1 M Tris, pH 7.4). Micrococcal nuclease was added to 4 pg/nil, and the mixture was incubated at 25" for 9 minutes. EDTA was added to 0.001 M and the remnant nuclei were centrifuged into a pellet. Fixation and electron microscopy was as in Fig. 1. Electron microscopy was by Elaine Lenk.
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The existence of this “quasi-stable” hnRNA population is strongly suggested by two observations. The amount of polyadenylylated molecules in the nucleus a t steady-state is far greater than would be expected on the basis of the purely short-lived hnRNA component, as shown below. Second, “chase” experiments conducted in the presence of high concentrations of glucosamine clearly indicate the presence of a multicomponent hnHNA population, a portion of which appears to decay with approxiniatcly a 20-minute half-life while the remainder has a half-life of about 100 minutes (see pp. 384-385). However, the glucosamine technique has a number of unanticipated pitfalls and is not completely understood at present. Therefore, we take, at present, the existence of the long-lived hnRNA component principally from the large steady-state content of polyadenylylatcd moleculcs.
A. mRNA Sequences in Steady-State hnRNA A major problem in the study of hnRNA has been the isolation of nuclear RNA free of cytoplasmic contamination. Total HcLa hnRNA prepared from nuclei washed using the double detergent procedure ( 6 ) remains contaminated with a small but significant amount of cytoplasmic species. This is concluded from the presence in the nuclear fraction of u p to 3%of the total cellular 18 S ribosomal RNA and thus, presumably, 3% of cellular polyribosomes. To reduce the cytoplasmic contamination of the hnRNA, an additional step was added to the cellular fractionation procedure. The nuclear structure can be disrupted by exposing the detergcnt-washed nuclei to high ionic strength (0.4 M ammonium sulfate) ( 7 ) . However, the bulk of the hnRNA remains associated with the chromatin and thus can be separated froin contaminating polyribosomes, which dissociate under these conditions. A small subfraction of hnRNA is released by the ammonium sulfate, but this accounts for very little of the steady-state material ( 7 ) . The hnRNA is then extracted with phenol/chloroform and extensively trcated with DNase ( 8). Nuclear RNA prepared in this way retains 1%( o r less) of the total cellular 18 S rHNA, most of which is probably nascent 18 S KNA in the nucleolus ( 6 ) . The presence of the ribonuclease inhibitors poly ( vinylsulfate ) , spermine and N-ethylmalcimide during the purification results in the isolation of very large hnRNA. More than half the molecules carrying poIy( A ) sediment faster than 45 S whcn isolated in this way ( Fig. 3a). Having achieved an extensive purification of hnRNA, we measured the relative cytoplasmic and nuclear p l y ( A) content of HeLa cells by hybridizing [3H]poly(U ) to each fraction ( 9 ) .In several different steadystate cytoplasmic and nuclear preparations, 20%of the cellular poly( A ) (by weight) is in nuclear RNA. [Less than one-fifth of the nuclear
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FIC. 3. Sedimentation distribution of purified nuclear RNA. Steady-state nuclear RNA was isolated from HeLa cells. The R N A was analyzed by sedimentation in a 15-302 sucrose gradient in dodecyl sulfate (0.1%)buffer. The gradient was assayed by hybridizing ["H]poly(U ) to a portion of each fraction. The locations of the 28 S, 18 S and 4 S R N A markers were taken from a parallel gradient. ( a ) Native hnRNA. Sedimentation was at 18 K rprn in the Spinco SW 41 rotor for 15 hours at 25°C. ( b ) Alkaline-cleaved, p l y ( A ) -containing hnRNA fragments. Native hnRNA was alkaline-cleaved for 15 minutes at 0°C. The poly( A)-containing fragments were isolated by poly( U ) -Scpharosc chromatography as described ( 23). Sedimentation was at 25 K rpm in the SW 41 rotor for 1G hours at 25°C. For details, see Herman ct al. ( 2 3 ) .
[3H]poly(U) binding is in the oligo(A) fraction.] This ratio of cytoplasmic to nuclear poly( A ) in HeLa cells is comparable to that obtained recently by Johnson et d.( 8 ) from growing mouse fibroblast 3T6 cells ( 2 : 1 ) , using both steady-state labeling with 3 2 P 0 , and [ 3 H ] p ~ I yU( ) hybridization. A cDNA copy of the 3' terminal of the purified steady-state HeLa hnRNA ( A,, ) was synthesized using avian myeloblastosis virus reverse transcriptase and oligo ( clT ) as primer. However, hnRNA contains short stretches of 30-50 adcnylatc residues [oligo( A) 1, which can be distinguished from the 3'-poly( A ) (approximately 200 AMP residues) by their internal positions in the molecules and by their transcription from the cellular DNA (10, 11 ). After limited alkaline hydrolysis of the hnRNA, the oligo ( A )-containing fragments were removed by differential affinity chromatography using poly( U)-Sepharose ( 9 ) ; the remaining poly ( A ) adjacent fragments are shown in Fig. 3b.
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LOG Rot
FIG. 4. Hybridization of cDNA transcribed from hnRNA( A,,) fragments compared to cDNA from mRNA. NLIC~CYU RNA(A.) fragments and niRNA(A,,) were purified, and cDNA was prepared from each as described ( 2 3 ) . RNA excess hybridizations were performed using a 1000-2000-fold excess of driver RNA. mRNA concentrations ( R 0 ) were calculated from the poly( A ) content of the preparation assuming that the poly( A ) is 4% of the chain length. Nuclear RNA concentrations were calculated as explained in the text. ( A , A ) nuclear cDNA driven by niRNA(A,,); ( X ) nuclear cDNA driven by hnRNA(A,) fragments; (0, 0 ) message cDNA drivcn by mRNA( A,,).
Figure 4 shows the results of an experiment in which the cDNA transcript of the cleaved nuclear RNA(A,,) was hybridized to an excess of mRNA( A,, ), The saturation value obtained in these hybridizations provides a measure of the fraction of hnRNA molccules sharing sequences with cytoplasmic mRNA. A t saturation, approximately 45-50% of the input nuclear cDNA hybridizes to thc mRNA(A,,). Figure 4 also shows that by log R,,t = 2, 67%of this same cDNA has hybridized to the hnRNA( A,,) fragments. Thus the nuclear cDNA anneals under similar conditions to a significantly greater extent with nuclear RNA than with cytoplasmic mRNA. The low saturation value achieved using cytoplasmic mRNA as “driver” is therefore due not to an inherent inability of the nuclear cDNA to hybridize, but rather to the presence of poly ( A ) -containing sequences that have no detectable counterparts in the cytoplasm. Thus at most 70% (46/67) of the nuclear cDNA transcribed from the 3’ terminus of the hnRNA( A,,) fragments is complementary to mRNA( A,). Assuming that reverse transcriptase copies RNA sequences in proportion to their relative abundance within the population, only 70%of the polyadenylylated HeLa
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nuclear RNA contains sequences at the 3' terminus related to those in mRNA ( A, ) . The most complex transition, extending from a log Rot of 0.7-2, contains approximately 33% of the cytoplasmic sequences but 65%of the hybridizable nuclear cDNA. If selective copying of the complex nuclear sequences has not occurred, these complex sequences constitute a relatively larger proportion of the nuclear RNA than of the cytoplasmic niRNA. Nevertheless, the actual number of RNA molecules in the nucleus containing the scarce sequences is lower than in the cytoplasm. It has been suggested that the largc hnRNA molecules result from the artifactual aggregation of smaller nuclear molecules. To show that mRNA sequences are in truly large molecules, we selected hnRNA molecules sedimenting faster than 45 S in an aqueous sucrose gradient, and then denatured these large molecules in Me,SO ( 1 2 ) . The fraction of these large molecules (20%)that still sedimented faster than 45 S in a 5 to 20% sucrose gradient in M e 3 0 was recovered and treated as outlined above to obtain poly ( A )-containing fragments. The hybridization of the cDNA prepared from this denatured, cleaved hnRNA( A,) is shown in Fig. 5. Approximately 40% (uncorrected) of this cDNA hybridizes to the mRNA( A,,). Kinetics of the hybridization are essentially identical to those for the hybridization of the cDNA prepared from the total cleaved hnRNA(A,) (Fig. 4).
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FIG.5 . Hybridization of cDNA transcribed from large hnRNA molecules. Nuclear RNA sedimenting faster than 45 S in an aqueous 15 to 30%sucrose gradient was isolated. This large RNA was denatured with MeSO and then centrifuged in a 5 to 20% sucrose gradient in MeSO at 40 K rpm for 20 hours in the Spinco SW 40 rotor. Those molecules again sedimenting faster than 45 S were pooled and alkali-cleaved, and the poly( A)-containing fragments were purified by poly( U ) -Sepharose chromatography. cDNA was prepared from the ( An)-containing fragments and then hybridized to an excess of mRNA( A"). Rot = concentration of RNA nucleotide x time.
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The results of the hybridization of nuclear cDNA to mRNA ( A,, ) suggest that at least a portion of the nuclear RNA molecules contain message sequences at their 3’ terminus adjacent to the poly( A). However, these experiments do not indicate how many of the cytoplasmic sequences are found in nuclear RNA. To answer this question, cDNA was prepared from the cytoplasmic mRNA(A,,) and annealed to a large excess of hnRNA( A,,). The cDNA from cytoplasmic mRNA hybridizes much more slowly to hnRNA than to its own template (Fig. 6 ) . This suggests that the rapidly hybridizing (abundant) sequences in the cytoplasm are much reduced in the nucleus relative to the scarce sequences. This agrees with the conclusions drawn from the hybridization of nuclear cDNA to cytoplasmic RNA ( Fig. 4 ) . An unambiguous interpretation of the hybridization of cytoplasmic cDNA to nuclear RNA requires that the contribution by cytoplasmic mRNA contamination be negligible. Most of the message sequences contaminating the nuclear RNA should be from the abundant classes. HeLa message cDNA was therefore separated into two fractions, one containing the transcripts of the abundant, and the other of the scarce, mRNA. Each cDNA fraction was then hybridized to the hnRNA separately. Fractionation was accomplished by annealing the HeLa message cDNA with mRNA( A,,) to an Rot value of approximately 1.5 and then separating the hybridized (abundant) from the unhybridized (scarce) message cDNA by chromatography on hydroxylapatite ( 1 3 ) . Separation was confirmed by the hybridization of each cDNA fraction to the mRNA (Fig. 6b). When the abundant message cDNA is annealed to cleaved, &go( dT)cellulose-purified hnRNA, hybridization occurs at values of Rut approximately 10 times those at which the same cDNA hybridizes to messenger RNA (Fig. 6 b ) . This displacement can be used to establish an absolute maximum level of cytoplasmic contamination of the nuclear RNA preparation (that is, 10%)by assuming, in the extreme, that all the observed hybridization of the abundant message cDNA is with cytoplasmic RNA contaminating the hnRNA. Similarly, the scarce message cDNA should hybridize 10-fold more slowly to nuclear RNA than to messenger RNA if only cytoplasmic contamination were driving the reaction. The data in Fig. 6 show that the scarce message cDNA hybridizes 4 times faster than was predicted by assuming 10% cytoplasmic contamination. This shows that most of the scarce mRNA sequences are present in hnRNA. The rate at which scarce message cDNA is driven into hybrid form by nuclear RNA is, however, 2.5-fold slower than when this cDNA is driven by messenger RNA. This is further evidence that the message sequences adjacent to poly( A) in the nucleus are diluted by poly( A)containing nonmessage sequences,
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FIG.6. Hybridization of HeLa “message” cDNA to hnRNA. ( a ) Total message cDNA. Unfractionated HeLa message cDNA was hybridized to an excess of cleaved, oligo( dT)-cellulose-bound hnRNA (X-X). [See Herman et al. ( 2 3 ) for an explanation of how nuclear RNA concentrations were determined.] The kinetics of hybridization of total message cDNA with mRNA(A,,) are reproduced from Fig. 4 for comparison ( ----). ( b ) Abundant and scarce message cDNA. Total HeLa message cDNA. Total HeLa message cDNA was annealed with mRNA(A.) to an Rat value of 1.5. The hybridized (abundant) cDNA was separated from the unhybridized ( scarce ) by chromatography on hydroxylapatite. Each fraction was then hybridized to an excess of mRNA( A,) or cleaved, oligo( dT)-cellulose-bound hnRNA: ( A--A) abundant cDNA driven by total mRNA(A,,); ( A-A) abundant cDNA driven by nuclear RNA; (.--a) scarce cDNA driven by total mRNA(A.); (0-0) scarce cDNA driven by nuclear RNA.
The number of copies of hnRNA molecules containing the scarce message sequences can be estimated from the data presented here. We have assumed that the HeLa cell has a total of 5 x lo5 mRNA molecules. One third of these are in the scarce class, which has a sequence complexity of -lo1. Thus there are about 15 copies of each scarce message
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per cell, Since 7%of the p l y ( A)-adjacent message sequences are in the nucleus and approximately 65%of these are “scarce” sequences, thcre is about 0.7 copy of each per nucleus. Considering the approximations used, it is very possible that the actual number is one copy per nucleus. Most important, there appear to be transcripts in the nuclcus corresponding to most or all of the active DNA regions.
B. Association of hnRNA with Other Structures To estimate the physical association of these hnRNA molecules with the nuclear skeleton and to chromatin, hnRNA was labelcd for 3 hours, the length of time sufficient to approach a steady state. We have already seen that most of this RNA is tightly associated with the chromatinnuclear skeleton complex that sediments after ammonium sulfate treatment. Part of this linkage is to the nuclear skeleton itself. Thc DNA of the chromatin can be almost quantitatively removed while the hnRNA remains associated with a rapidly sedimcnting structure with properties of the remnant nuclear skeleton. The data in Table I indicate that after removal of over 95% of nuclear DNA, at least 80%of hnRNA remains associated with the nuclear skeleton even after ammonium sulfate fraction. The double-stranded regions of the hnRNA are apparently involved in the linkage of these molecules to the nuclear skeleton. Dige5tion of isolated nuclei with pancreatic ribonuclease removes upward of 80%of hnRNA but leaves 50-80% of the double-stranded regions intact and attached to the nuclear skeleton chromatin complex. The data in Table I show the retention of the protected double-strand pieces and their relative resistance to subsequent elution by ammonium sulfate. Removal of most of the chromatin with DNase at the same time as RNase digestion has very little effect on the final result, either with respect to double-strand yield or the resistance to ammonium sulfate. The results suggest that a significant portion of the double-strand loops are firmly attached to the nuclear skeleton, but not to the chromatin to a significant degree, at least by the criteria applied here. A very similar result is obtained when the poly(A) segment of hnRNA is examined. The results in Table I show also that the poly( A ) scgment remains attachcd to the nuclear skeleton as long as it is covalently linked to the hnHNA molecules and the chromatin remains intact. In parallel to the double strands, the digestion of the chromatin with DNase and the digestion of the hnRNA with RNase docs not result in the extensive liberation of the poly(A) segment. Thus it appears that thc attachment of poly(A) also does not require the integrity of chromatin and suggests that this segment is, in fact, affixed to the nuclear superstructure.
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IIeLa cells were labeled overnight with [I4C]thymidine(Part A only) and for 3 hours with [311]uridineor [3H]adcnosinc. Thc cclls were broken in low ionic strength buffer (0.01 hf NaCI, 0.01 hI Tris, pH 7.4, 1.5 mM MgCI?) by the addition of NP-40 to 1 %. Isolated nuclei were trcated, as indicated, in the same buffer with pancreatic IZNase (10 pg/tnl) and pancreatic 1)Nase (120 gg/xnl) for 10 minutes at 25°C. Nuclei were fractionated by resuspension in 0.4 M (NH4)2S04as descrihed previously ( 7 ) . Fractions were cxtractcd with phenol and digested with R.Nasc a t high ionic strength (0.25 hl NaCI, 0.01 M MgC12, 0.01 M Tris, p H 7.4) and assayed for double-stranded IlNA content by clcctrophorrsis on 14 % polyacrylamidc gcls or for poly(A) content by electrophoresis on 10 % gels.
The linkage is apparently a relatively firm one since ribonuclease followed by ammonium sulfate leaves the poly(A) segment with the skeleton. The picture that emerges from these results is of relatively long-lived HNA transcripts attached to the nuclear skeleton by both the doublestranded RNA regions and the 3’ poly( A) tails. The double strands and poly(A) segment are not necessarily the only sites of attachment of hnRNA to subcellular structures. Rather, these are portions that are resistant to ribonuclease digestion and thus their location in the nuclear skeleton-chromatin complex is easily determined. We suggest that these transcripts play a role in organizing chromatin and keeping active or euchromatin in its native state. This hypothesis would be consistent with
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the observation that chromatin undcrgoes significant reorganization after the inhibition of RNA metabolism with a drug such as actinomycin. We therefore suggest that there are two classes of hnRNA, one that is short-lived and serves, at least in part, as the precursor to cytoplasmic mRNA, and a second class that is relativcly long-livcd and contains long transcripts terminating in poly ( A ) , These long-lived transcripts appear to be attached to the nuclear skeleton and perhaps also to the chromatin. A most plausible assumption would be that the sites of attachment are related to the region producing the quasi-stable transcript. Since these regions are active, one would conclude that transcription continues in the region where the quasi-stable hnRNA is attached. This raises the possibility that the transcript that is not used for morphological purposes is not of the same size or from exactly the same region as thc quasi-stable transcript. Certainly there are suggestions that message may arise from the 5’ end of hnRNA as indicated by conservation of 5’ caps’ from the nucleus to the cytoplasm ( Perry, private communication ) . Nevertheless, all the cytoplasmic mRNA appears to be present in sequences a t the 3’ ends of the quasi-stable transcripts. There is also an observation of the subfraction of hnRNA consisting of smaller than average nuclear transcripts ( although still considerably larger than cytoplasmic mRNA) that behaves as a major precursor to cytoplasmic mcssage ( 7 ) . This subfraction was removed in present procedures, and yet the remaining hnRNA still contains most if not all of the sequences of cytoplasmic mRNA. Such pieces of evidence are merely tantalizing and certainly do not prove that a particular transcription site may have more than one size or type of transcript. However, the data presented here do suggest a possible structural role for the quasi-stable hnRNA molecules and raise the possibility of several types of transcripts with different functions at a given locus.
111. Low-Molecular-Weight RNA Species In this section we turn to another form of gene expression in eukaryotic cells and use the word “message” in a broader sense than simply a sequence coding for polypeptides. I t was established nearly 10 years ago that there are low-molecular-wcight RNA molecules found in the nucleus and, more recently, in the cytoplasm of the cells of higher organisms that d o not serve any role in protein synthesis (references to much of the earlier work are given in Table 11). These molecules have no known
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CELLULAR SKELETONS AND
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analogy in prokaryotic HNA metabolism and are among the things that clearly distinguish cukaryotic RNA metabolism from that of lower organisms. Since their initial discovery and characterization, the work on these RNA species has been rather desultory since no very plausible proposal has been made concerning their possible function. Our recent results indicate that these low-molecular-weight RNA species, found in very specific subcellular locations, appear to be involved in cellular structure. In particular, a number of species are specifically associated with the nuclear skeleton. Of the two cytoplasmic species, one at least appears, in part, to be associated with membranes and appears in oncornaviruses derived from mammalian cells. What follows is a listing of the small, stable nuclear and cytoplasmic RNAs and a brief description of what is presently known about the metabolism and localization of each.
A. SnA The position of SnA relative to SiiB and SnC is altered in this gel system compared to the electrophoresis system used previously (14).Its identification, however, is unambiguous owing to its being located exclusively in the nucleolar fraction ( a s shown in lane 3 of Fig. 7). It also remains in the nucleus after the release of chromatin in the form of
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FIG. 7. Identification of the small-molecular-weight RNAs in the HeLa cell. Electrophoresis in 6 to 15%dodecyl sulfate polyacrylaniide slab gels. Each fraction was prepared from 2 x lo6 cells labeled for 16 hours with 5 pCi of ['H]uridine per milliliter under normal growth conditions. 1, Nuclear fraction; 2, Cytoplasmic fraction; 3, nucleolar fraction. For details, see Zieve and Penman ( 2 4 ) .
nucleosomes ( n u bodies2). Labeled SnA is not found associated with the nucleolus until 15 minutes after the addition of the label to the cells. During the first 15 minutes of labeling, SnA is found transiently in the cytoplasmic fraction. Thus for a brief period after its synthesis, SnA is not fixed in the nucleolus, and either goes through an initial cytoplasmic stage before entering the nucleolus or leaks into the cytoplasm during fractionation. After 2 hours of continuous labeling, equal amounts of SnA are found in the cytoplasmic and nuclear fractions. After a l6-hour label, >go% of the SnA in the cells is found in the nucleolus. 'See papers by Axel and by Paoletti et al. in this volume.
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Several results suggest that SnA is involved in nucleolar processing. A fraction of SnA is hydrogen-bonded to 32-28 S nucleolar intermediates, as first reportcd by Prestayko et al. (15). After 6 hours of labeling, we find 5% of the SnA in the cell hydrogen-bonded to nucleolar 32-28 S material. If these cells are allowed to grow for an additional 12 hours or longer, 30%of the total cellular SiiA is found hydrogen-bonded to the nucleolar material. SnA is never found hydrogen-bonded to any cytoplasmic material. Significant amounts of SnA (25%)are still synthesized in the presence of 0.04 pglml of actinomycin D, a concentration that is sufficient totally to inhibit ribosomal precursor formation (16). In addition, the SnA synthesized in the presence of low levels of actinomycin is found only in the cytoplasmic fraction of the cell. A similar result is found if cells are labeled with an RNA precursor in the presence of 5-fluorouridine, an inhibitor of ribosomal processing (17, 18). No SnA is found in the cell if cells are labeled for 16 hours in the presence of 5-fluorouridine. The fixation of SnA in the nucleus and its stability in the cell appear to be dependent upon normal nucleolar functioning.
B. SnB, SnC These species are associated predominantly with the nuclear skeleton. In our gel system, they run very close to each other, C having only a slightly faster mobility. When cells labeled for 16 hours are fractionated into nucleus and cytoplasm, between 25%and 40%of the labeled species SnB and SnC appear in the cytoplasmic fraction (Fig. 7). All the SnB that remains in the nuclei is found in the nucleoplasmic fraction. Most of the SnC that remains in the nucleus is also found in the nucleoplasmic fraction; however, between 5% and 10%is found in the nucleolar preparations. When chromatin is released from the nuclei by enzymic digestion, all the SnB found in the nucleus pellets with the nuclear skeleton. A small amount of SnC is released from the nuclei by both micrococcal and pancreatic DNase digestions.
C. SnD SnD is the most abundant of the small RNA species found in the nuclei of mammalian cells. In the cold fractionation procedure, up to 10%of this species is found in the cytoplasmic fraction, a t least some attributable to mitotic cells (19). ( O n our gels, SnD often runs as a curved band, as can be seen in the gel patterns.) It is the only small species released from the nucleus during brief warming as, for example, occurs during the digestion for nu bodies. The SnD released is in very
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large structures that sediment at 40 S and contain a large number of distinct polypeptidcs.
D. SnF, SnH These species are the least abundant of the major small nuclear RNAs, and their behavior has not been exhaustively studied. Both are found only in the nucleoplasm. SnF is partially released from the nuclei by micrococcal nuclease digestion but not hy the pancreatic DNase, which suggests that the RNase activity is required to detach it from the nuclear skeleton. SnH is not released from the nuclei by either of the enzyme digestions. SnH has a half-life of 30 hours; however, SnH disappears in the presence of high levels of actinomycin D.
E. SnG’ SnG’ is a stable molecule associated with the nucleoplasm. I t was originally identified as a form of cytoplasmic 5 S RNA but was later shown to be a methylated 5 S RNA distinct from cytoplasmic ribosomal 5 S (20). Its electrophoretic mobility is slightly different from cytoplasmic 5 S on our gels, and a recent report shows that a nuclear 5 S RNA actually has a base sequence very different from that of cytoplasmic 5 S (21 ).
F. SnK SiiK is the only specics that has not been definitely localized. It appears to varying degrees in both nucleus and cytoplasm. After a 10-minute period of labeling, radioactive SnK is found totally in the cytoplasmic fraction. After 90 minutes of labeling, about 10%of it becomes associated with the nucleus. After 18 hours of labeling, the distribution of SnK is quite variable and in different experiments ranged from 60%cytoplasmic/ 40%nuclear to 95%cytoplasmic/ 5% nuclear. The most interesting aspect of the metabolism of SnK is its migration into thc nuclear fraction after exposure of the cells to high levels of actiiiomycin D. When prelabeled cells are treated with 5 pg/ml of actinomycin D, increasing ilmounts of SnK become associated with the nuclear fraction, and by 4 hours 90%of the prelabeled SnK is found in the nucleus. This behavior could not be induced by any other inhibitor investigated. Unlike the other small nuclear species, S I X is not methylated (25). W e have confirmed this and have also found that species K is metabolically stable. In addition, the synthesis of SnK is the most resistant to actinomycin D of all the small RNAs, with the exception of 5 S rRNA and tRNA. At 0.1 pg/inl actinomycin D, SnK is still synthcsized at 40%of the control rate.
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G.SnP SnP is a stable species, found only in the nucleus, which we tentatively identify as a new low-molecular-weight HNA. SnP is obvious in Fig. 7. This RNA often runs as a very diffuse band in the aqueous gels. It becomes labeled very slowly, and its behavior has not been studied extcnsively.
H.
S c l or Oncornavirus 7 S RNA This RNA species has been previously identified as a nuclear species (20). In our fractionation, it is totally cytoplasmic and is the small cytoplasmic RNA that has recently been described as occurring in cells and in the virion of oncornaviruses (22). It was also reported that ScL is associated with polyribosomes. W e have been unable to reproduce this result using either hypotonic or isotonic lysis buffers. More than 90%of ScL sediments more slowly than ribosomal subunits in a sucrose gradient when detergent lysis is used to fractionate the cells. A significant portion of ScL is associated with membranes, A membrane fraction contains 30% of the ScL of a mechanically prepared cytoplasmic extract. If the membrane fraction is prepared from cytoplasm cxposed to EDTA, the amount of ScL is reduced to 15%.If membranes are dissolved with 0.5%deoxycholate and 0.5%Brij 58, none of the ScL is found in rapidly scdimenting structures. I t therefore seems probable that ScL is, in part, found in membranes, and it can be concluded that this portion is truly cytoplasmic in localization. The ScL RNA from HeLa cells and from oncornavirus are probably identical molecules ( Fig. 8 ) . Both have the same electrophoretic mobility for the main band, and each has a family of conformers that run more rapidly than the principal band, Conformers have been isolated from gels and rerun in urea. Under denaturing conditions, the conformers from both the cells and the virus migrate with the same mobility, which is identical to that of the main band of ScL. In addition, ScL from both cell and virus can be shifted in mobility by heating prior to electrophoresis. The data in Fig. 8 show that both viral and cellular RNA is shifted in mobility to a single band whose migration rate is intermediate between the originaI main band and the fastest running conformers. The numbcr of properties the viral and cellular RNA molecules have in common is sufficient to suggest their identity at least with respect to physical behavior. Indeed, it seems very possible that the molecule found in the virion is of cellular origin. The presence of this molecule in membranes suggests that it may become associated with the virion during the process of maturation and budding.
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FIG. 8. Equivalent mobility of ScL and viral 7 S R N A under nondenaturing and denattiring conditions. Electrophoresis was carried out in a 6 to 15%gradient slab gel as described ( 2 4 ) . 1, Cytoplasmic fraction from 6 x loa cells labeled for 2 hours; 2, isolated ScL; 3, isolated ScL heated for 7 minutes at 70°C in sample buffer before application to the gel; 4, MLV-M RNA; 5, MLV-M R N A heated for 7 minutes at 70°C in sample buffer before application to the gel.
ScL resembles SnK in that it is not methylated and is metabolically stable. Its synthesis is inhibited 801 by 0.1 &ml actinoniycin D. The results of this survey are summarized in Table I1 and Fig. 9 together with the nomenclaturc of othcr workers. The specific subcellular location suggests that these H N A species are involved in cellular structure either as passive participants in subcellular organization or as active directors of architectural construction. More recent studies have gone further and show that the cytoplasmic species K and L are highly enriched in preparations of the cytoplasmic “skeleton,” further supporting the notion that these RNA molecules arc always found in association with specific cellular structures. The role of these small HNA molecules is only a subject of speculation a t present, but it may be noted that they represent a flow of information
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FIG. 9. Localizations of the small RNA species of the HeLa cell. The schematic diagram of the HeLa cell shows the locations of the small cytoplasmic ( S c ) and small nuclear ( S n ) RNA species in the cytoplasm, nucleoplasm and nucleolus ( n u ) .
from the genome that does not go through the process of protein synthesis. Thus especially with regard to cellular organization, we must consider the possibility that there arc forms of gene expression in eukaryotes that affect cell behavior directly and not through the mediation of protein synthesis. The amount of such information may be rather small, if, in fact, we have completely described all the small R N A species. If, however, many of the “confonners” scem in the electrophoretic pattern should prove to be molecules of different base sequence, the amount of genetic information flowing through this pathway could be considerable indeed.
IV. Summary This report presents experimental results that suggest an interrelation between cellular topology and nucleic acid metabolism. Gentle lysis of HeLa cells with a nonionic detergent reveals an elaborate remnant structure that retains much of the cellular morphology. Electron micrographs and biochemical studies show that a majority of the protein synthetic apparatus remains affixed to this “cytoskeleton.” The nature and even the reality of this attachment remains to be elucidated. However, the suggestion that protein synthesis takes place in a topologically ordered manner would help explain numerous observations showing that polyribosomes appear not to be randomly distributed in the cytoplasm of cells. Thus, the skeleton structure possibly plays an important role in cytoplasmic R N A metabolism. Two of the small R N A species found in mammalian cells, ScK and ScL, are intimately associated with the cytoskeleton, and ScL is, in part, associated with membranes. The function of these molecules is unknown, but there is the possibility that they serve a structural role and may help determine the architecture of the cytoskeleton. If so, then R N A metabolism could play a direct role in determining cellular topology.
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The nucleus also has a “skeletal” structure, although much less can be visualized in this case. We have suggested that hnRNA molecules are attached by both their double-stranded segments and 3’ poly( A ) tails to this nuclear framcwork. This suggests a possible structural role for these transcripts in keeping chromatin properly organized in the interphase nucleus. Also, many of the small HNA species are found localized in this nuclear skeleton, suggesting that they also may play a roIe in either structure or function determination. The experiments described here, while not definitive, indicate an aspect of the metazoan cell with no obvious counterpart in bacteria, that is, an interrelation between spatial organization and RNA metabolism.
ACKNOWLEDGMENTS We would like to thank Carol Hahnfeld and Laura Ransom for their excellent technical assistance. This work was supported by grants from the National Institutes of Health (NIH 2 R 0 1 CA08416; NIH CA12174) and from the National Science Foundation (BMS 73 06859). R. Herman is a recipient of a National Institutes of Health postdoctord fellowship ( 6 F22 DEOl655). J. Willian~s was a Harkness Foundation Fellow. G. Zieve is the recipient of a predoctoral fellowship from the National Science Foundation.
REFERENCES 1. M. L. Shelanski and H. Feif, in “Structure and Function in the Nervous System” (C. Bourne, ed.), Vol. 6, pp. 47-80. Academic Press, New York, 1972. 2. R. V. Rice and A. C. Brady, CSHSQB 37, 439 (1972). 3. R. P. Goldman and D. M. Knipe, CSHSQB 37,523 (1972). 4. S. Yen, D. Dahl, M. Schachmer and M. L. Shelanski, PNAS 73, 529 (1976). 5. R. Berezney and D. Coffey, BBRC 60, 1410 (1974). 6. S . Penman, J M B 17, 177 (1966). 7 . R. Price, L. Ransom and S. Penman, Cell 2, 253 ( 1974). 8. L. F. Johnson, J. G. Williams, H. T. Abelson, H. Green and S. Penman, Cell 4, 69 (1975). 9. J. 0. Bishop, M. Rosbash and D. Evans, J M B 85, 75 (1974). 10. G. R. Molloy, W. Jelinek, M. Salditt and J. E. Darnell, Cell 1, 43 (1974). 11. H. Nakazato, M. Edmonds and B. W. Kopp, PNAS 71, 200 (1974). 12. E. Derman and J. E. Darnell, Cell 3, 255 ( 1974). 13. J. G. Williams and S. Penman, Cell 6 , 197 (1975). 14. R. Weinberg and S . Penman, J M B 38, 289 (1968). 15. A. W. Prestayko, M. Tonato and H. Busch, J M B 47, 505 (1970). 16. S. Penman, C. Vesco and M. Penman, J M B 39, 49 (1968). 17. D. S. Wilkinson and H. C. Pitot, JBC 248, 63 (1973). 18. B. Brdr, D.B. Rifkin and E. Reich, FEBS Lett. 24,345 ( 1972). 19. A. Rein, B B A 232, 306 (1970). 20. R. Weinberg and S . Penman, B B A 190, 10 ( 1969). 21. T. S. Ro-Choi and H. Busch, in “The Cell Nucleus” ( H . Busch, ed.), 3rd ed., pp. 151-208. Academic Press, New York, 1974.
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22. T. A. Walker, N. R. Pace, R. L. Erikson, E. Erikson and F. Behr, PNAS 71, 3390 ( 1974). 23. R. C. Herman, J. G. Williams and S. Penman, Cell 7,429 (1976). 24. G. Zieve and S. Penman, Cell 8, 19 (1976). 25. S. Frederiksen, I. R. Pederson, P. Hellung-Larsen and J. Engberg, BBA 340, 64 (1974). 26. hl. F. Marzluff, E. L. White, R. Benjamin and R. C . Huang, Chromatin Biochem. 14, 3715 (1975).
Progress in the study of eukaryotic, and especially metazoan, cell biology is quite apparent from the contributions at this conference. Nevertheless, the challenge remains to elucidate those properties of gene expression, presumably through RNA metabolism, that serve to make a metazoan animal the complex arrangement of biological materials that it is. In particular, morphogenesis involves a bewildering variety of cell growth, movement, changes in architecture and the development of special biochemical pathways. Although the impressive work on the RNA metabolism of higher organisms has established profound differences from the metabolism of prokaryotes, so far, with few exceptions, little relates our studies to the obvious problems of metazoan biology. In this report, we describe some of our first tentative efforts to relate RNA metabolism to the unique properties of a mctazoan cell. W e present suggestive evidence that the architecture of the metazoan cell, in this case mammalian, is intimately involved with RNA metabolism.
1. Cytoplasmic Skeleton We describe two preparations (“skeletons”) from HeLa cells, one cytoplasmic and one nuclear. [The term “skeleton” is currently used to suggest a number of different cellular structures; perhaps in the near future a more definitive terminology will be adopted.] The choice of HeLa cells for a study of cell architecture may seem odd since these cells have little in the way of morphologically distinct features. Nevertheless, for other reasons, this work was started using this rather nondescript workhorse of a cultured cell, and a quite remarkable degree of internal structure was found. 379
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Figure 1 is a low-power clectron micrograph of a HeLa cell gently lysed with Triton X-100 in an isotonic buffer. The general structure of a cell and nucleus are clearly visible, and even such specialized morphological entities as surface microspikes seem to be partially preserved. What is remarkable about this preparation is how few of the cellular constituents remain. The procedure extracts most of the phospholipids and a11 of the soluble components, such as proteins and transfer RNA, and mitochondria1 constituents have apparently been leached out. Furthermore, the cold extraction procedure breaks down microtubules, and tubulin is quantitatively removed. Actin filaments are not visible; actin is found largely in extracted proteins either because of its unpolymerized state in these cells or owing to an instability of nonmuscle f-actin under these extraction conditions. What remains is comprised of a network of filaments that at higher magnification are seen to be the intcrincdiate filaments previously described ( 1-3). These apparently interconnect and possibly mesh with the as yet undefined proteins, which appear condensed in rather diffuse blotches. Thus, a major component of the structure that maintains morphology in the absence of microtubules or microfilaments appears to be the intermediate filaments. The analysis of “skeleton” proteins shows that the 53,000-dalton subunit of these filaments described by Shelanski and co-workers ( 4 ) is, in fact, a major component and is quantitatively rctained in the preparation. Most s t a r t h g is the retention of most of the active polyribosomes of the cell. These are quite apparent in the electron micrograph, and biochemical measurements show that at least 75% of the active cellular polyribosomes remain attached by some linkage to the cytoskeleton. In contrast to the active polyribosomes, the inactive monomers are largely extracted by the lysis procedure. The polyribosomes always appear to be associated with the blotches of condensed protein apparent in the cellular network. Where the intermediate fibers are particularly dense, the polyribosomes appear excluded. The most difficult thing to establish at this point is whether the association of polyribosomes with the cytoskeleton represents a truc in viuo state or is some artifact of the extraction procedure. The major evidence that this association may represent the true distribution of polyribosomes in the intact cell is the observation that ribosomal monomers do not stick to the “skeleton” to any significant degree. Of course, the active ribosomes may very well have components that lead to an artifactual association; at present, this possibility cannot be ruled out. Experiments are in progress to determine whether the products of the extracted polyribosomes are in any way differentfrom those that remain attached to the skeleton.
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FIG. 1. Cytaskeleton of HeLa cells. HeLa cells in suspension culture were harvested, washed and resuspended in isotonic, low ionic strength buffer (0.25 M sucrose, 0.01 M NaCI, 0.003 M MgCI,, 0.01 M Tris, pH 7.4) containing 1%Triton X-100. The cells were vortexed briefly and centrifuged into a pellet. Fixation was with 2.5% glutaraldehyde followed by 1%osmium tetroxide. The sample was dehydrated in alcohol, embedded in Epon-Araldite and sectioned. Micrographs were taken with a JEM lOOB at 80 Kev. The microscopy was carried out by Elaine Lenk.
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Taken at face value, and assuming that the preparation is not plagued by artifacts, the electron-microscope and biochemical studies suggest strongly that the protein synthetic machineiy of the cell does not float about freely but, even in the case of “free” (as opposed to membranebound) polyribosomes, is localized within the cytoplasm. This would serve to explain such puzzling morphological observations as the apparent exclusion of ribosomes from certain regions of the cell, such as the vicinity of the centriole, Also, the localization of the protein synthetic machinery on relatively spatially stable structures makes some teleological sense. In such cases as cell division or extensive ccll movement, a randomly diffusing protein synthetic system would be out of cellular control and the partition of polyribosomes between daughter cells or parts of extended cell would be left to chance. Also, there may be situations similar to those involving products of membrane-bound polyribosomes, where the spatial location of polyribosome products is important. All this is speculative and requires considerable further effort to demonstrate the reality of the suggested topological control of the proteinsynthesizing components of the mammalian cell.
II. The Nuclear Skeleton and hnRNA We return to the cytoskeleton below, but consider here another “skeleton”, this time the one associated with the nucleus and presumably related to the structures previously described by Berezney and Coffey ( 5). Figure 2 shows an early preparation in which about SM of the DNA has been removed by microccocal nuclease digestion. Much of the remaining DNA appears localized in the perinuclear heterochromatin, which appears to be relatively less accessible to nuclease. Later preparatory techniques, for which electron micrographs are not presently available, remove the remaining plasma membrane components surrounding the nuclear shell and more than 95% of the total nuclear DNA. Even in this early electron micrograph (Fig. 2 ) , it is possible to see that the nucleus retains its shape (somewhat distorted in this preparation by high centrifugation forces) and that suggestions of internal structure are visible through the empty space left by the removed chromatin. A prominent nucleolus is visible; it is possible that it is simply trapped by the nuclcar shell. We postulate a relation between nuclear metabolism, chromatin organization, and this nuclear skclcton. This interrelation is apparently accomplished by a class of large hnRNA molecules that are relatively long-lived, many terminating with poly( A ) , and that appear to be attached to both the skeleton and the chromatin.
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FIG.2 . Nuclear skeleton of HeLa Cells. HeLa cells were broken in hypotonic buffer (0.01 M NaCl, 0.003 M MgCl,, 0.01 M Tris, pH 7.4) to which 1%Triton X-100 was added. Nuclei were separated by centrifugation and resuspended in digestion buffer ( 5 % sucrose, 10.' CaCl?, 0.1 M Tris, pH 7.4). Micrococcal nuclease was added to 4 pg/nil, and the mixture was incubated at 25" for 9 minutes. EDTA was added to 0.001 M and the remnant nuclei were centrifuged into a pellet. Fixation and electron microscopy was as in Fig. 1. Electron microscopy was by Elaine Lenk.
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The existence of this “quasi-stable” hnRNA population is strongly suggested by two observations. The amount of polyadenylylated molecules in the nucleus a t steady-state is far greater than would be expected on the basis of the purely short-lived hnRNA component, as shown below. Second, “chase” experiments conducted in the presence of high concentrations of glucosamine clearly indicate the presence of a multicomponent hnHNA population, a portion of which appears to decay with approxiniatcly a 20-minute half-life while the remainder has a half-life of about 100 minutes (see pp. 384-385). However, the glucosamine technique has a number of unanticipated pitfalls and is not completely understood at present. Therefore, we take, at present, the existence of the long-lived hnRNA component principally from the large steady-state content of polyadenylylatcd moleculcs.
A. mRNA Sequences in Steady-State hnRNA A major problem in the study of hnRNA has been the isolation of nuclear RNA free of cytoplasmic contamination. Total HcLa hnRNA prepared from nuclei washed using the double detergent procedure ( 6 ) remains contaminated with a small but significant amount of cytoplasmic species. This is concluded from the presence in the nuclear fraction of u p to 3%of the total cellular 18 S ribosomal RNA and thus, presumably, 3% of cellular polyribosomes. To reduce the cytoplasmic contamination of the hnRNA, an additional step was added to the cellular fractionation procedure. The nuclear structure can be disrupted by exposing the detergcnt-washed nuclei to high ionic strength (0.4 M ammonium sulfate) ( 7 ) . However, the bulk of the hnRNA remains associated with the chromatin and thus can be separated froin contaminating polyribosomes, which dissociate under these conditions. A small subfraction of hnRNA is released by the ammonium sulfate, but this accounts for very little of the steady-state material ( 7 ) . The hnRNA is then extracted with phenol/chloroform and extensively trcated with DNase ( 8). Nuclear RNA prepared in this way retains 1%( o r less) of the total cellular 18 S rHNA, most of which is probably nascent 18 S KNA in the nucleolus ( 6 ) . The presence of the ribonuclease inhibitors poly ( vinylsulfate ) , spermine and N-ethylmalcimide during the purification results in the isolation of very large hnRNA. More than half the molecules carrying poIy( A ) sediment faster than 45 S whcn isolated in this way ( Fig. 3a). Having achieved an extensive purification of hnRNA, we measured the relative cytoplasmic and nuclear p l y ( A) content of HeLa cells by hybridizing [3H]poly(U ) to each fraction ( 9 ) .In several different steadystate cytoplasmic and nuclear preparations, 20%of the cellular poly( A ) (by weight) is in nuclear RNA. [Less than one-fifth of the nuclear
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FIC. 3. Sedimentation distribution of purified nuclear RNA. Steady-state nuclear RNA was isolated from HeLa cells. The R N A was analyzed by sedimentation in a 15-302 sucrose gradient in dodecyl sulfate (0.1%)buffer. The gradient was assayed by hybridizing ["H]poly(U ) to a portion of each fraction. The locations of the 28 S, 18 S and 4 S R N A markers were taken from a parallel gradient. ( a ) Native hnRNA. Sedimentation was at 18 K rprn in the Spinco SW 41 rotor for 15 hours at 25°C. ( b ) Alkaline-cleaved, p l y ( A ) -containing hnRNA fragments. Native hnRNA was alkaline-cleaved for 15 minutes at 0°C. The poly( A)-containing fragments were isolated by poly( U ) -Scpharosc chromatography as described ( 23). Sedimentation was at 25 K rpm in the SW 41 rotor for 1G hours at 25°C. For details, see Herman ct al. ( 2 3 ) .
[3H]poly(U) binding is in the oligo(A) fraction.] This ratio of cytoplasmic to nuclear poly( A ) in HeLa cells is comparable to that obtained recently by Johnson et d.( 8 ) from growing mouse fibroblast 3T6 cells ( 2 : 1 ) , using both steady-state labeling with 3 2 P 0 , and [ 3 H ] p ~ I yU( ) hybridization. A cDNA copy of the 3' terminal of the purified steady-state HeLa hnRNA ( A,, ) was synthesized using avian myeloblastosis virus reverse transcriptase and oligo ( clT ) as primer. However, hnRNA contains short stretches of 30-50 adcnylatc residues [oligo( A) 1, which can be distinguished from the 3'-poly( A ) (approximately 200 AMP residues) by their internal positions in the molecules and by their transcription from the cellular DNA (10, 11 ). After limited alkaline hydrolysis of the hnRNA, the oligo ( A )-containing fragments were removed by differential affinity chromatography using poly( U)-Sepharose ( 9 ) ; the remaining poly ( A ) adjacent fragments are shown in Fig. 3b.
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LOG Rot
FIG. 4. Hybridization of cDNA transcribed from hnRNA( A,,) fragments compared to cDNA from mRNA. NLIC~CYU RNA(A.) fragments and niRNA(A,,) were purified, and cDNA was prepared from each as described ( 2 3 ) . RNA excess hybridizations were performed using a 1000-2000-fold excess of driver RNA. mRNA concentrations ( R 0 ) were calculated from the poly( A ) content of the preparation assuming that the poly( A ) is 4% of the chain length. Nuclear RNA concentrations were calculated as explained in the text. ( A , A ) nuclear cDNA driven by niRNA(A,,); ( X ) nuclear cDNA driven by hnRNA(A,) fragments; (0, 0 ) message cDNA drivcn by mRNA( A,,).
Figure 4 shows the results of an experiment in which the cDNA transcript of the cleaved nuclear RNA(A,,) was hybridized to an excess of mRNA( A,, ), The saturation value obtained in these hybridizations provides a measure of the fraction of hnRNA molccules sharing sequences with cytoplasmic mRNA. A t saturation, approximately 45-50% of the input nuclear cDNA hybridizes to thc mRNA(A,,). Figure 4 also shows that by log R,,t = 2, 67%of this same cDNA has hybridized to the hnRNA( A,,) fragments. Thus the nuclear cDNA anneals under similar conditions to a significantly greater extent with nuclear RNA than with cytoplasmic mRNA. The low saturation value achieved using cytoplasmic mRNA as “driver” is therefore due not to an inherent inability of the nuclear cDNA to hybridize, but rather to the presence of poly ( A ) -containing sequences that have no detectable counterparts in the cytoplasm. Thus at most 70% (46/67) of the nuclear cDNA transcribed from the 3’ terminus of the hnRNA( A,,) fragments is complementary to mRNA( A,). Assuming that reverse transcriptase copies RNA sequences in proportion to their relative abundance within the population, only 70%of the polyadenylylated HeLa
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nuclear RNA contains sequences at the 3' terminus related to those in mRNA ( A, ) . The most complex transition, extending from a log Rot of 0.7-2, contains approximately 33% of the cytoplasmic sequences but 65%of the hybridizable nuclear cDNA. If selective copying of the complex nuclear sequences has not occurred, these complex sequences constitute a relatively larger proportion of the nuclear RNA than of the cytoplasmic niRNA. Nevertheless, the actual number of RNA molecules in the nucleus containing the scarce sequences is lower than in the cytoplasm. It has been suggested that the largc hnRNA molecules result from the artifactual aggregation of smaller nuclear molecules. To show that mRNA sequences are in truly large molecules, we selected hnRNA molecules sedimenting faster than 45 S in an aqueous sucrose gradient, and then denatured these large molecules in Me,SO ( 1 2 ) . The fraction of these large molecules (20%)that still sedimented faster than 45 S in a 5 to 20% sucrose gradient in M e 3 0 was recovered and treated as outlined above to obtain poly ( A )-containing fragments. The hybridization of the cDNA prepared from this denatured, cleaved hnRNA( A,) is shown in Fig. 5. Approximately 40% (uncorrected) of this cDNA hybridizes to the mRNA( A,,). Kinetics of the hybridization are essentially identical to those for the hybridization of the cDNA prepared from the total cleaved hnRNA(A,) (Fig. 4).
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FIG.5 . Hybridization of cDNA transcribed from large hnRNA molecules. Nuclear RNA sedimenting faster than 45 S in an aqueous 15 to 30%sucrose gradient was isolated. This large RNA was denatured with MeSO and then centrifuged in a 5 to 20% sucrose gradient in MeSO at 40 K rpm for 20 hours in the Spinco SW 40 rotor. Those molecules again sedimenting faster than 45 S were pooled and alkali-cleaved, and the poly( A)-containing fragments were purified by poly( U ) -Sepharose chromatography. cDNA was prepared from the ( An)-containing fragments and then hybridized to an excess of mRNA( A"). Rot = concentration of RNA nucleotide x time.
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The results of the hybridization of nuclear cDNA to mRNA ( A,, ) suggest that at least a portion of the nuclear RNA molecules contain message sequences at their 3’ terminus adjacent to the poly( A). However, these experiments do not indicate how many of the cytoplasmic sequences are found in nuclear RNA. To answer this question, cDNA was prepared from the cytoplasmic mRNA(A,,) and annealed to a large excess of hnRNA( A,,). The cDNA from cytoplasmic mRNA hybridizes much more slowly to hnRNA than to its own template (Fig. 6 ) . This suggests that the rapidly hybridizing (abundant) sequences in the cytoplasm are much reduced in the nucleus relative to the scarce sequences. This agrees with the conclusions drawn from the hybridization of nuclear cDNA to cytoplasmic RNA ( Fig. 4 ) . An unambiguous interpretation of the hybridization of cytoplasmic cDNA to nuclear RNA requires that the contribution by cytoplasmic mRNA contamination be negligible. Most of the message sequences contaminating the nuclear RNA should be from the abundant classes. HeLa message cDNA was therefore separated into two fractions, one containing the transcripts of the abundant, and the other of the scarce, mRNA. Each cDNA fraction was then hybridized to the hnRNA separately. Fractionation was accomplished by annealing the HeLa message cDNA with mRNA( A,,) to an Rot value of approximately 1.5 and then separating the hybridized (abundant) from the unhybridized (scarce) message cDNA by chromatography on hydroxylapatite ( 1 3 ) . Separation was confirmed by the hybridization of each cDNA fraction to the mRNA (Fig. 6b). When the abundant message cDNA is annealed to cleaved, &go( dT)cellulose-purified hnRNA, hybridization occurs at values of Rut approximately 10 times those at which the same cDNA hybridizes to messenger RNA (Fig. 6 b ) . This displacement can be used to establish an absolute maximum level of cytoplasmic contamination of the nuclear RNA preparation (that is, 10%)by assuming, in the extreme, that all the observed hybridization of the abundant message cDNA is with cytoplasmic RNA contaminating the hnRNA. Similarly, the scarce message cDNA should hybridize 10-fold more slowly to nuclear RNA than to messenger RNA if only cytoplasmic contamination were driving the reaction. The data in Fig. 6 show that the scarce message cDNA hybridizes 4 times faster than was predicted by assuming 10% cytoplasmic contamination. This shows that most of the scarce mRNA sequences are present in hnRNA. The rate at which scarce message cDNA is driven into hybrid form by nuclear RNA is, however, 2.5-fold slower than when this cDNA is driven by messenger RNA. This is further evidence that the message sequences adjacent to poly( A) in the nucleus are diluted by poly( A)containing nonmessage sequences,
KNA
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LOG R o t
FIG.6. Hybridization of HeLa “message” cDNA to hnRNA. ( a ) Total message cDNA. Unfractionated HeLa message cDNA was hybridized to an excess of cleaved, oligo( dT)-cellulose-bound hnRNA (X-X). [See Herman et al. ( 2 3 ) for an explanation of how nuclear RNA concentrations were determined.] The kinetics of hybridization of total message cDNA with mRNA(A,,) are reproduced from Fig. 4 for comparison ( ----). ( b ) Abundant and scarce message cDNA. Total HeLa message cDNA. Total HeLa message cDNA was annealed with mRNA(A.) to an Rat value of 1.5. The hybridized (abundant) cDNA was separated from the unhybridized ( scarce ) by chromatography on hydroxylapatite. Each fraction was then hybridized to an excess of mRNA( A,) or cleaved, oligo( dT)-cellulose-bound hnRNA: ( A--A) abundant cDNA driven by total mRNA(A,,); ( A-A) abundant cDNA driven by nuclear RNA; (.--a) scarce cDNA driven by total mRNA(A.); (0-0) scarce cDNA driven by nuclear RNA.
The number of copies of hnRNA molecules containing the scarce message sequences can be estimated from the data presented here. We have assumed that the HeLa cell has a total of 5 x lo5 mRNA molecules. One third of these are in the scarce class, which has a sequence complexity of -lo1. Thus there are about 15 copies of each scarce message
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per cell, Since 7%of the p l y ( A)-adjacent message sequences are in the nucleus and approximately 65%of these are “scarce” sequences, thcre is about 0.7 copy of each per nucleus. Considering the approximations used, it is very possible that the actual number is one copy per nucleus. Most important, there appear to be transcripts in the nuclcus corresponding to most or all of the active DNA regions.
B. Association of hnRNA with Other Structures To estimate the physical association of these hnRNA molecules with the nuclear skeleton and to chromatin, hnRNA was labelcd for 3 hours, the length of time sufficient to approach a steady state. We have already seen that most of this RNA is tightly associated with the chromatinnuclear skeleton complex that sediments after ammonium sulfate treatment. Part of this linkage is to the nuclear skeleton itself. Thc DNA of the chromatin can be almost quantitatively removed while the hnRNA remains associated with a rapidly sedimcnting structure with properties of the remnant nuclear skeleton. The data in Table I indicate that after removal of over 95% of nuclear DNA, at least 80%of hnRNA remains associated with the nuclear skeleton even after ammonium sulfate fraction. The double-stranded regions of the hnRNA are apparently involved in the linkage of these molecules to the nuclear skeleton. Dige5tion of isolated nuclei with pancreatic ribonuclease removes upward of 80%of hnRNA but leaves 50-80% of the double-stranded regions intact and attached to the nuclear skeleton chromatin complex. The data in Table I show the retention of the protected double-strand pieces and their relative resistance to subsequent elution by ammonium sulfate. Removal of most of the chromatin with DNase at the same time as RNase digestion has very little effect on the final result, either with respect to double-strand yield or the resistance to ammonium sulfate. The results suggest that a significant portion of the double-strand loops are firmly attached to the nuclear skeleton, but not to the chromatin to a significant degree, at least by the criteria applied here. A very similar result is obtained when the poly(A) segment of hnRNA is examined. The results in Table I show also that the poly( A ) scgment remains attachcd to the nuclear skeleton as long as it is covalently linked to the hnHNA molecules and the chromatin remains intact. In parallel to the double strands, the digestion of the chromatin with DNase and the digestion of the hnRNA with RNase docs not result in the extensive liberation of the poly(A) segment. Thus it appears that thc attachment of poly(A) also does not require the integrity of chromatin and suggests that this segment is, in fact, affixed to the nuclear superstructure.
CELLULAR SKELETONS AND
Assocl.\rrIoiv
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H N R N A WITH
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TABLE I NuCLfi:.\It
SK1.:LI.;TON . \N D
THE
A. Ilistribution of hnKNA( %) Control
+DNase
hnItNA DNA
hnIlNA DNA
lleleasrd from nucleus llelrased i n (NH4)2S04 Prllct
1
1
)
4
94
87
9*j
Control ~
Iteleased from nucleus Rcleased in (NH4),S04 Pellet
9 5 86
70 26 4
4 9
B. Ilistribution of 1)ouhlc-Stranded RNA( %)
~_+IlNase
+
RNasc +L)Nase
___
15 37 48
5 .55 40
C . Ihtribution of Poly(A)( %) Control ~
lleleased from nucleus Iteleased in (NH4)2S04 Pellet
CHROMATINa
4 15 XI
+ RNase ____ 23 10 67
+DNase flZNase
~
29 16 55
IIeLa cells were labeled overnight with [I4C]thymidine(Part A only) and for 3 hours with [311]uridineor [3H]adcnosinc. Thc cclls were broken in low ionic strength buffer (0.01 hf NaCI, 0.01 hI Tris, pH 7.4, 1.5 mM MgCI?) by the addition of NP-40 to 1 %. Isolated nuclei were trcated, as indicated, in the same buffer with pancreatic IZNase (10 pg/tnl) and pancreatic 1)Nase (120 gg/xnl) for 10 minutes at 25°C. Nuclei were fractionated by resuspension in 0.4 M (NH4)2S04as descrihed previously ( 7 ) . Fractions were cxtractcd with phenol and digested with R.Nasc a t high ionic strength (0.25 hl NaCI, 0.01 M MgC12, 0.01 M Tris, p H 7.4) and assayed for double-stranded IlNA content by clcctrophorrsis on 14 % polyacrylamidc gcls or for poly(A) content by electrophoresis on 10 % gels.
The linkage is apparently a relatively firm one since ribonuclease followed by ammonium sulfate leaves the poly(A) segment with the skeleton. The picture that emerges from these results is of relatively long-lived HNA transcripts attached to the nuclear skeleton by both the doublestranded RNA regions and the 3’ poly( A) tails. The double strands and poly(A) segment are not necessarily the only sites of attachment of hnRNA to subcellular structures. Rather, these are portions that are resistant to ribonuclease digestion and thus their location in the nuclear skeleton-chromatin complex is easily determined. We suggest that these transcripts play a role in organizing chromatin and keeping active or euchromatin in its native state. This hypothesis would be consistent with
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the observation that chromatin undcrgoes significant reorganization after the inhibition of RNA metabolism with a drug such as actinomycin. We therefore suggest that there are two classes of hnRNA, one that is short-lived and serves, at least in part, as the precursor to cytoplasmic mRNA, and a second class that is relativcly long-livcd and contains long transcripts terminating in poly ( A ) , These long-lived transcripts appear to be attached to the nuclear skeleton and perhaps also to the chromatin. A most plausible assumption would be that the sites of attachment are related to the region producing the quasi-stable transcript. Since these regions are active, one would conclude that transcription continues in the region where the quasi-stable hnRNA is attached. This raises the possibility that the transcript that is not used for morphological purposes is not of the same size or from exactly the same region as thc quasi-stable transcript. Certainly there are suggestions that message may arise from the 5’ end of hnRNA as indicated by conservation of 5’ caps’ from the nucleus to the cytoplasm ( Perry, private communication ) . Nevertheless, all the cytoplasmic mRNA appears to be present in sequences a t the 3’ ends of the quasi-stable transcripts. There is also an observation of the subfraction of hnRNA consisting of smaller than average nuclear transcripts ( although still considerably larger than cytoplasmic mRNA) that behaves as a major precursor to cytoplasmic mcssage ( 7 ) . This subfraction was removed in present procedures, and yet the remaining hnRNA still contains most if not all of the sequences of cytoplasmic mRNA. Such pieces of evidence are merely tantalizing and certainly do not prove that a particular transcription site may have more than one size or type of transcript. However, the data presented here do suggest a possible structural role for the quasi-stable hnRNA molecules and raise the possibility of several types of transcripts with different functions at a given locus.
111. Low-Molecular-Weight RNA Species In this section we turn to another form of gene expression in eukaryotic cells and use the word “message” in a broader sense than simply a sequence coding for polypeptides. I t was established nearly 10 years ago that there are low-molecular-wcight RNA molecules found in the nucleus and, more recently, in the cytoplasm of the cells of higher organisms that d o not serve any role in protein synthesis (references to much of the earlier work are given in Table 11). These molecules have no known
’ Re caps, see articles in Part I of
this volume.
CELLULAR SKELETONS AND
RNA
Alternative nomenclaturcsa*h
A B C 1) E F
US
Ilalflifec.“ c 1
U?
c 2
UIB
C3
5.8 S
UI,
.i S I, I1 6 s 111 4.5 8 I, 11, 111
G
11
K
c4
CS
C6
“\Yral 7 S”
L
393
MESSAGES
Stable 20 Hr 2
Localization Nucl~olar Nuclear skeleton Nuclear skeleton Nuclear Ribosomal Nucleoplasmic Ribosomal Nu c1ear Nuclear skclcton Cytoplasmic and nuclear Cytoplasmic /membrane
110-Choi and Busch ( 2 1 ) . al. ( 2 2 ) . c Weinberg and Penman (20). (1 Frederiksen ct al. ( 2 5 ) . e hlarzluff ct al. (26).
* Walker rt
analogy in prokaryotic HNA metabolism and are among the things that clearly distinguish cukaryotic RNA metabolism from that of lower organisms. Since their initial discovery and characterization, the work on these RNA species has been rather desultory since no very plausible proposal has been made concerning their possible function. Our recent results indicate that these low-molecular-weight RNA species, found in very specific subcellular locations, appear to be involved in cellular structure. In particular, a number of species are specifically associated with the nuclear skeleton. Of the two cytoplasmic species, one at least appears, in part, to be associated with membranes and appears in oncornaviruses derived from mammalian cells. What follows is a listing of the small, stable nuclear and cytoplasmic RNAs and a brief description of what is presently known about the metabolism and localization of each.
A. SnA The position of SnA relative to SiiB and SnC is altered in this gel system compared to the electrophoresis system used previously (14).Its identification, however, is unambiguous owing to its being located exclusively in the nucleolar fraction ( a s shown in lane 3 of Fig. 7). It also remains in the nucleus after the release of chromatin in the form of
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RONALD HERMAN ET AL.
FIG. 7. Identification of the small-molecular-weight RNAs in the HeLa cell. Electrophoresis in 6 to 15%dodecyl sulfate polyacrylaniide slab gels. Each fraction was prepared from 2 x lo6 cells labeled for 16 hours with 5 pCi of ['H]uridine per milliliter under normal growth conditions. 1, Nuclear fraction; 2, Cytoplasmic fraction; 3, nucleolar fraction. For details, see Zieve and Penman ( 2 4 ) .
nucleosomes ( n u bodies2). Labeled SnA is not found associated with the nucleolus until 15 minutes after the addition of the label to the cells. During the first 15 minutes of labeling, SnA is found transiently in the cytoplasmic fraction. Thus for a brief period after its synthesis, SnA is not fixed in the nucleolus, and either goes through an initial cytoplasmic stage before entering the nucleolus or leaks into the cytoplasm during fractionation. After 2 hours of continuous labeling, equal amounts of SnA are found in the cytoplasmic and nuclear fractions. After a l6-hour label, >go% of the SnA in the cells is found in the nucleolus. 'See papers by Axel and by Paoletti et al. in this volume.
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Several results suggest that SnA is involved in nucleolar processing. A fraction of SnA is hydrogen-bonded to 32-28 S nucleolar intermediates, as first reportcd by Prestayko et al. (15). After 6 hours of labeling, we find 5% of the SnA in the cell hydrogen-bonded to nucleolar 32-28 S material. If these cells are allowed to grow for an additional 12 hours or longer, 30%of the total cellular SiiA is found hydrogen-bonded to the nucleolar material. SnA is never found hydrogen-bonded to any cytoplasmic material. Significant amounts of SnA (25%)are still synthesized in the presence of 0.04 pglml of actinomycin D, a concentration that is sufficient totally to inhibit ribosomal precursor formation (16). In addition, the SnA synthesized in the presence of low levels of actinomycin is found only in the cytoplasmic fraction of the cell. A similar result is found if cells are labeled with an RNA precursor in the presence of 5-fluorouridine, an inhibitor of ribosomal processing (17, 18). No SnA is found in the cell if cells are labeled for 16 hours in the presence of 5-fluorouridine. The fixation of SnA in the nucleus and its stability in the cell appear to be dependent upon normal nucleolar functioning.
B. SnB, SnC These species are associated predominantly with the nuclear skeleton. In our gel system, they run very close to each other, C having only a slightly faster mobility. When cells labeled for 16 hours are fractionated into nucleus and cytoplasm, between 25%and 40%of the labeled species SnB and SnC appear in the cytoplasmic fraction (Fig. 7). All the SnB that remains in the nuclei is found in the nucleoplasmic fraction. Most of the SnC that remains in the nucleus is also found in the nucleoplasmic fraction; however, between 5% and 10%is found in the nucleolar preparations. When chromatin is released from the nuclei by enzymic digestion, all the SnB found in the nucleus pellets with the nuclear skeleton. A small amount of SnC is released from the nuclei by both micrococcal and pancreatic DNase digestions.
C. SnD SnD is the most abundant of the small RNA species found in the nuclei of mammalian cells. In the cold fractionation procedure, up to 10%of this species is found in the cytoplasmic fraction, a t least some attributable to mitotic cells (19). ( O n our gels, SnD often runs as a curved band, as can be seen in the gel patterns.) It is the only small species released from the nucleus during brief warming as, for example, occurs during the digestion for nu bodies. The SnD released is in very
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RONALD HERMAN ET AL.
large structures that sediment at 40 S and contain a large number of distinct polypeptidcs.
D. SnF, SnH These species are the least abundant of the major small nuclear RNAs, and their behavior has not been exhaustively studied. Both are found only in the nucleoplasm. SnF is partially released from the nuclei by micrococcal nuclease digestion but not hy the pancreatic DNase, which suggests that the RNase activity is required to detach it from the nuclear skeleton. SnH is not released from the nuclei by either of the enzyme digestions. SnH has a half-life of 30 hours; however, SnH disappears in the presence of high levels of actinomycin D.
E. SnG’ SnG’ is a stable molecule associated with the nucleoplasm. I t was originally identified as a form of cytoplasmic 5 S RNA but was later shown to be a methylated 5 S RNA distinct from cytoplasmic ribosomal 5 S (20). Its electrophoretic mobility is slightly different from cytoplasmic 5 S on our gels, and a recent report shows that a nuclear 5 S RNA actually has a base sequence very different from that of cytoplasmic 5 S (21 ).
F. SnK SiiK is the only specics that has not been definitely localized. It appears to varying degrees in both nucleus and cytoplasm. After a 10-minute period of labeling, radioactive SnK is found totally in the cytoplasmic fraction. After 90 minutes of labeling, about 10%of it becomes associated with the nucleus. After 18 hours of labeling, the distribution of SnK is quite variable and in different experiments ranged from 60%cytoplasmic/ 40%nuclear to 95%cytoplasmic/ 5% nuclear. The most interesting aspect of the metabolism of SnK is its migration into thc nuclear fraction after exposure of the cells to high levels of actiiiomycin D. When prelabeled cells are treated with 5 pg/ml of actinomycin D, increasing ilmounts of SnK become associated with the nuclear fraction, and by 4 hours 90%of the prelabeled SnK is found in the nucleus. This behavior could not be induced by any other inhibitor investigated. Unlike the other small nuclear species, S I X is not methylated (25). W e have confirmed this and have also found that species K is metabolically stable. In addition, the synthesis of SnK is the most resistant to actinomycin D of all the small RNAs, with the exception of 5 S rRNA and tRNA. At 0.1 pg/inl actinomycin D, SnK is still synthcsized at 40%of the control rate.
CELLULAR SKELETONS AND
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397
G.SnP SnP is a stable species, found only in the nucleus, which we tentatively identify as a new low-molecular-weight HNA. SnP is obvious in Fig. 7. This RNA often runs as a very diffuse band in the aqueous gels. It becomes labeled very slowly, and its behavior has not been studied extcnsively.
H.
S c l or Oncornavirus 7 S RNA This RNA species has been previously identified as a nuclear species (20). In our fractionation, it is totally cytoplasmic and is the small cytoplasmic RNA that has recently been described as occurring in cells and in the virion of oncornaviruses (22). It was also reported that ScL is associated with polyribosomes. W e have been unable to reproduce this result using either hypotonic or isotonic lysis buffers. More than 90%of ScL sediments more slowly than ribosomal subunits in a sucrose gradient when detergent lysis is used to fractionate the cells. A significant portion of ScL is associated with membranes, A membrane fraction contains 30% of the ScL of a mechanically prepared cytoplasmic extract. If the membrane fraction is prepared from cytoplasm cxposed to EDTA, the amount of ScL is reduced to 15%.If membranes are dissolved with 0.5%deoxycholate and 0.5%Brij 58, none of the ScL is found in rapidly scdimenting structures. I t therefore seems probable that ScL is, in part, found in membranes, and it can be concluded that this portion is truly cytoplasmic in localization. The ScL RNA from HeLa cells and from oncornavirus are probably identical molecules ( Fig. 8 ) . Both have the same electrophoretic mobility for the main band, and each has a family of conformers that run more rapidly than the principal band, Conformers have been isolated from gels and rerun in urea. Under denaturing conditions, the conformers from both the cells and the virus migrate with the same mobility, which is identical to that of the main band of ScL. In addition, ScL from both cell and virus can be shifted in mobility by heating prior to electrophoresis. The data in Fig. 8 show that both viral and cellular RNA is shifted in mobility to a single band whose migration rate is intermediate between the originaI main band and the fastest running conformers. The numbcr of properties the viral and cellular RNA molecules have in common is sufficient to suggest their identity at least with respect to physical behavior. Indeed, it seems very possible that the molecule found in the virion is of cellular origin. The presence of this molecule in membranes suggests that it may become associated with the virion during the process of maturation and budding.
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RONALD HERMAN ET AL.
FIG. 8. Equivalent mobility of ScL and viral 7 S R N A under nondenaturing and denattiring conditions. Electrophoresis was carried out in a 6 to 15%gradient slab gel as described ( 2 4 ) . 1, Cytoplasmic fraction from 6 x loa cells labeled for 2 hours; 2, isolated ScL; 3, isolated ScL heated for 7 minutes at 70°C in sample buffer before application to the gel; 4, MLV-M RNA; 5, MLV-M R N A heated for 7 minutes at 70°C in sample buffer before application to the gel.
ScL resembles SnK in that it is not methylated and is metabolically stable. Its synthesis is inhibited 801 by 0.1 &ml actinoniycin D. The results of this survey are summarized in Table I1 and Fig. 9 together with the nomenclaturc of othcr workers. The specific subcellular location suggests that these H N A species are involved in cellular structure either as passive participants in subcellular organization or as active directors of architectural construction. More recent studies have gone further and show that the cytoplasmic species K and L are highly enriched in preparations of the cytoplasmic “skeleton,” further supporting the notion that these RNA molecules arc always found in association with specific cellular structures. The role of these small HNA molecules is only a subject of speculation a t present, but it may be noted that they represent a flow of information
CELLULAR SKELETONS AND
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hlESSAGES
399
Sc D
FIG. 9. Localizations of the small RNA species of the HeLa cell. The schematic diagram of the HeLa cell shows the locations of the small cytoplasmic ( S c ) and small nuclear ( S n ) RNA species in the cytoplasm, nucleoplasm and nucleolus ( n u ) .
from the genome that does not go through the process of protein synthesis. Thus especially with regard to cellular organization, we must consider the possibility that there arc forms of gene expression in eukaryotes that affect cell behavior directly and not through the mediation of protein synthesis. The amount of such information may be rather small, if, in fact, we have completely described all the small R N A species. If, however, many of the “confonners” scem in the electrophoretic pattern should prove to be molecules of different base sequence, the amount of genetic information flowing through this pathway could be considerable indeed.
IV. Summary This report presents experimental results that suggest an interrelation between cellular topology and nucleic acid metabolism. Gentle lysis of HeLa cells with a nonionic detergent reveals an elaborate remnant structure that retains much of the cellular morphology. Electron micrographs and biochemical studies show that a majority of the protein synthetic apparatus remains affixed to this “cytoskeleton.” The nature and even the reality of this attachment remains to be elucidated. However, the suggestion that protein synthesis takes place in a topologically ordered manner would help explain numerous observations showing that polyribosomes appear not to be randomly distributed in the cytoplasm of cells. Thus, the skeleton structure possibly plays an important role in cytoplasmic R N A metabolism. Two of the small R N A species found in mammalian cells, ScK and ScL, are intimately associated with the cytoskeleton, and ScL is, in part, associated with membranes. The function of these molecules is unknown, but there is the possibility that they serve a structural role and may help determine the architecture of the cytoskeleton. If so, then R N A metabolism could play a direct role in determining cellular topology.
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RONALD HERMAN ET AL.
The nucleus also has a “skeletal” structure, although much less can be visualized in this case. We have suggested that hnRNA molecules are attached by both their double-stranded segments and 3’ poly( A ) tails to this nuclear framcwork. This suggests a possible structural role for these transcripts in keeping chromatin properly organized in the interphase nucleus. Also, many of the small HNA species are found localized in this nuclear skeleton, suggesting that they also may play a roIe in either structure or function determination. The experiments described here, while not definitive, indicate an aspect of the metazoan cell with no obvious counterpart in bacteria, that is, an interrelation between spatial organization and RNA metabolism.
ACKNOWLEDGMENTS We would like to thank Carol Hahnfeld and Laura Ransom for their excellent technical assistance. This work was supported by grants from the National Institutes of Health (NIH 2 R 0 1 CA08416; NIH CA12174) and from the National Science Foundation (BMS 73 06859). R. Herman is a recipient of a National Institutes of Health postdoctord fellowship ( 6 F22 DEOl655). J. Willian~s was a Harkness Foundation Fellow. G. Zieve is the recipient of a predoctoral fellowship from the National Science Foundation.
REFERENCES 1. M. L. Shelanski and H. Feif, in “Structure and Function in the Nervous System” (C. Bourne, ed.), Vol. 6, pp. 47-80. Academic Press, New York, 1972. 2. R. V. Rice and A. C. Brady, CSHSQB 37, 439 (1972). 3. R. P. Goldman and D. M. Knipe, CSHSQB 37,523 (1972). 4. S. Yen, D. Dahl, M. Schachmer and M. L. Shelanski, PNAS 73, 529 (1976). 5. R. Berezney and D. Coffey, BBRC 60, 1410 (1974). 6. S . Penman, J M B 17, 177 (1966). 7 . R. Price, L. Ransom and S. Penman, Cell 2, 253 ( 1974). 8. L. F. Johnson, J. G. Williams, H. T. Abelson, H. Green and S. Penman, Cell 4, 69 (1975). 9. J. 0. Bishop, M. Rosbash and D. Evans, J M B 85, 75 (1974). 10. G. R. Molloy, W. Jelinek, M. Salditt and J. E. Darnell, Cell 1, 43 (1974). 11. H. Nakazato, M. Edmonds and B. W. Kopp, PNAS 71, 200 (1974). 12. E. Derman and J. E. Darnell, Cell 3, 255 ( 1974). 13. J. G. Williams and S. Penman, Cell 6 , 197 (1975). 14. R. Weinberg and S . Penman, J M B 38, 289 (1968). 15. A. W. Prestayko, M. Tonato and H. Busch, J M B 47, 505 (1970). 16. S. Penman, C. Vesco and M. Penman, J M B 39, 49 (1968). 17. D. S. Wilkinson and H. C. Pitot, JBC 248, 63 (1973). 18. B. Brdr, D.B. Rifkin and E. Reich, FEBS Lett. 24,345 ( 1972). 19. A. Rein, B B A 232, 306 (1970). 20. R. Weinberg and S . Penman, B B A 190, 10 ( 1969). 21. T. S. Ro-Choi and H. Busch, in “The Cell Nucleus” ( H . Busch, ed.), 3rd ed., pp. 151-208. Academic Press, New York, 1974.
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22. T. A. Walker, N. R. Pace, R. L. Erikson, E. Erikson and F. Behr, PNAS 71, 3390 ( 1974). 23. R. C. Herman, J. G. Williams and S. Penman, Cell 7,429 (1976). 24. G. Zieve and S. Penman, Cell 8, 19 (1976). 25. S. Frederiksen, I. R. Pederson, P. Hellung-Larsen and J. Engberg, BBA 340, 64 (1974). 26. hl. F. Marzluff, E. L. White, R. Benjamin and R. C . Huang, Chromatin Biochem. 14, 3715 (1975).