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The small molecular weight monodisperse nuclear RNA's in mitotic cells
306
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96780
T H E SMALL MOLECULAR W E I G H T M O N O D I S P E R S E N U C L E A R RNA's IN MITOTIC CELLS
ALAN R E I N Bionetics Research Laboratories, Inc., 7300 Pearl St., Bethesda, Md. 2ooi 4 (U.S.A.)
(Received September 7th, 197o )
SUMMARY
I. Since the small molecular weight monodisperse nuclear RNA's (snRNA's) are permanently associated with the nuclei of growing cells, they must be re-incorporated into the daughter nuclei after each cell division. Their state was investigated in H e L a cells arrested in metaphase with colcemide. When cells were fractionated at low ionic strength, the snRNA's were found to be in structures sedimenting over a wide range, but principally between 30 and 18o S. Different snRNA's were distributed differently over this size range. 2. A reconstruction experiment indicated that this sedimentation distribution was not the result of aggregation of free snRNA's with other celhllar materials during cell fractionation. 3- The structures containing the snRNA's in mitotic cells sediment much more slowly in gradients containing 0.3 M NaC1 plus 0.03 M MgC12 than in the low ionic strength medium mentioned above. 4. The results suggest that the snRNA's are carried in relatively small particles during mitosis. These particles m a y also exist in interphase nuclei; their salt lability is compatible with this possibility.
INTRODUCTION
It has recently been discovered that the nuclei of vertebrate cells contain, in addition to the ribosomal precursor RNA's and the nuclear heterodisperse RNA ~, a third class of RNA species. This class consists of nine RNA species, each of discrete molecular weight, ranging from 3 " 104 to 8 • lO 4. They are extensively methylated and metabolically quite stable. They are found only in the cell nucleus. They have now been reported in a variety of tissue culture lines 2-6 and in embryonic 3 and adult 7 tissues. Their functions are completely unknown. They will be referred to here as the "small molecular weight monodisperse nuclear R N A ' s " (snRNA's). When an exponentially growing cell population is labeled briefly with an RNA precursor and then grown in nonradioactive medium, the labeled snRNA's are found to remain associated with the nuclei over several cell generations 3. They must, therefore, re-associate with the nucleus after each division. This re-association Abbreviation: snRNA, small molecular weight monodisperse nuclear RNA. I3iochim. Biophys. Acta, 232 (I97 I) 3o6-313
SMALL NUCLEAR
RNA
IX MITOTIC CELLS
307
has in fact been demonstrated directly in synchronized cultures 8. In an a t t e m p t to understand the mechanism of this specific association with the nucleus after mitosis, we were prompted to investigate the state of these RNA's during metaphase. In particular, one possibility was that the RNA's are directed into the daughter nuclei b y being attached to the chromosomes. The results to be presented indicate that, rather, the snRNA's are contained in much smaller particles in mitotic cells. MATERIALS AND METHODS
Buffers used included o.oi M NaCl-o.ooI5 M MgC12-o.oI M Tris buffer, p H 7.4 (RSB buffer); 0. 5 M NaCl-o.o 5 M MgCl~-o.oI M Tris buffer, p H 7.4 (HSB buffer); and o.I M NaCl-o.ooI M EDTA-o.5 ~o sodium dodecyl sulfate-o.oI M "Iris buffer, p H 7.4 (SDS buffer). [5-zHlUridine (25 C/mmole) was purchased from New England Nuclear Corp. Thymidine was purchased from Calbiochem. All experiments were done with spinner cultures of H e L a type S 3 cells. The procedures for growing and synchronizing these cells, and for extraction and analysis of the snRNA's, were as described b y WEINBERG AND PENMAN3,8. Briefly, cells are grown in Joklik-modified Eagle's medium at a concentration of 2 • lO5-4 • lO 5 cells/ mh Populations of metaphase cells are obtained by synchronizing the cells b y double thymidine block and then collecting the cells in mitosis in colcemide. Cells were fractionated 9 at o °. RNA's were extracted with phenol and chloroform ~in the presence of sodium dodecyl sulfate and EDTA. Extractions were performed at room temperature, since at higher temperatures the 28-S associated RNA 1° is liberated from 28-S ribosomal RNA and interferes with the electrophoretic analysis of snRNA's in mitosis. Control experiments have shown that extractions at room temperature and at 55 ° yield the same amount of snRNA's from interphase cells. All cell fractions were treated with DNase after phenol extraction 3, and then electrophoresed on IO °/o polyacrylamide gels at 7 V/cm. The gels were then cut into I-ram slices and each slice was digested with N H 4 0 H and counted in a liquid scintillation counter. All gels contained 14C-labeled marker RNA as well as the 3H-labeled RNA being analyzed. These markers were an important aid in identifying the individual RNA peaks with confidence; however, for simplicity, they have been omitted from all but one of the figures. Although nine species of snRNA's, namely A, B, C, D, F, G', H, K, and L, have been described in H e L a cells8, only A, C, and D will be studied here. The other species are either present in too small amounts, or have electrophoretic mobilities too close to the cytoplasmic species (i.e. 5-S ribosomal RNA and transfer RNA) to allow analysis in mitotic cells. The partition of cellular DNA into the different cell fractions was analyzed b y measuring the radioactivity which remained trichloroacetic acid precipitable after dilution in SDS buffer and incubation overnight at 37 ° in 0.3 M NaOH. In long labeling periods such as those employed in these experiments, cells which are synthesizing DNA convert a considerable fraction of the [5-3H]uridine label into Es-SH]deoxycytidine and incorporate it into DNA. Mitotic indices were determined under the phase contrast microscope. The cells were first centrifuged and resuspended in RSB buffer to increase the visibility of the nucleus in nonmitotic cells. Biochim. Biophys. Acta, 232 (1971) 3o6-313
308
A. REIN
RESULTS
The first experiment to be presented is a single centrifugation fractionation oi prelabeled mitotic cells blocked in metaphase with colcemide. A culture of HeLa cells was labeled with [3H]uridine between the first and second thymidine blocks. As is shown in Fig. Ia, the culture was divided into two parts upon release from the second thymidine block. One was treated with colcemide for the first 7 h after release, and then harvested; none of the cells in this portion had yet entered mitosis. The other was treated with colcemide from 7 to 14. 5 h after release, so that when it was harvested 80 % of the cells were in metaphase. The two suspensions were homogenized and fractionated by differential centrifugation, and each fraction was analyzed for DNA and the snRNA's (Figs. Ib-If). In the interphase culture, the nuclear pellet contained nearlv, all the snRNA's and 93 O/;oof the DNA. The control shows that colcemide treatment per se does not liberate snRNA's from large nucleoprotein structures. In the mitotic culture, however, the snRNA's were found mainly in the postmitochondrial supernatant, which contained only 5 % of the DNA. (Although 20 % of the cells had visible nuclear membranes, the "nuclear" pellet with 7 ° % of the DNA, contained no detectable snRNA. It seems likely that the snRNA's in the mitochondrial pellet were derived from the remaining interphase cells. These cells were evidently not proceeding into mitosis (Fig. Ia) and perhaps had particularly fragile nuclei. Comparison of the yields of the snRNA's from the two cultures shows that the RNA's were completely recovered from the mitotic cells. l~er~b~ CelN:C¥ 0 q l 0 s m
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a Fig'. i. s n R N A ' s in d i f f e r e n t cell f r a c t i o n s of i n t e r p h a s e a n d m i t o t i c cells. 2 • lO7 cells in i o o ml of m e d i u m were t r e a t e d w i t h 2 mM t h y m i d i n e for 15 h a n d t h e n c e n t r i f u g e d a n d r e s u s p e n d e d in i o o ml of fresh m e d i u m . 15o/zC of t S H l u r i d i n e were a d d e d 6. 5 h later, a n d 2 mlV[ t h y m i d i n e w a s a d d e d 2 h a f t e r t h e label. The cells were a g a i n c e n t r i f u g e d a n d r e s u s p e n d e d in IOO ml of fresh m e d i u m a f t e r 14 h in t h y m i d i n e . Colcemide (0.6/~g/ml} w a s a d d e d t o o n e - t h i r d of t h e c u l t u r e u p o n r e s u s p e n s i o n in fresh m e d i u m . 7 h l a t e r t h e s e cells were h a r v e s t e d a n d c o l c e m i d e a d d e d to t h e r e m a i n d e r of t h e culture. The h a r v e s t e d cells were r i n s e d in E a r l e ' s saline, i n c u b a t e d for i o rain a t o ° in R S B buffer a n d b r o k e n w i t h a D o u n c e h o m o g e n i z e r . N u c l e i w e re p e l l e t e d b y cent r i f u g a t i o n a t i o o o × g for 3 rain a n d d i g e s t e d b y d e o x y r i b o n u c l e a s e in H S B buffer. 7.5 h l a t e r t h e r e m a i n d e r of t h e c u l t u r e w a s f r a c t i o n a t e d b y e x a c t l y t h e s a m e proc e dure , e x c e p t t h a t a second c e n t r i f u g a t i o n of 12 ooo × g for i o rain was p e r f o r m e d on t h e s u p e r n a t a n t of t h e fi rs t c e n t r i f u g a tion. A l i q u o t s of all cell f r a c t i o n s were r e m o v e d for d e t e r m i n a t i o n of r a d i o a c t i v i t y i n D N A , a n d RBTA's were e x t r a c t e d a n d e l e c t r o p h o r e s e d for 8 h. (a) M i t o t i c indices of t h e c o l c e m i d e - c o n t a i n i n g c u l t u r e s . (b) N u c l e a r p e l l e t of t h e i n t e r p h a s e cells. (c) C y t o p l a s m of t h e i n t e r p h a s e cells. (d) " N u c l e a r " p e l l e t of t h e m i t o t i c cells. (e) 12 ooo × g p e l l e t of t h e m i t o t i c cells (f) 12 ooo × g supern a t a n t of t h e m i t o t i c cells. u
Biochim. Biophys. Acta, 232 (I971) 3o6-313
SMALL NUCLEAR
RNA
309
IN MITOTIC CELLS
An experiment was then performed to determine the size of the structures containing the snRNA's in mitotic cells. A culture was labeled with [3H~uridine during synchronization and the cells were collected in mitosis with colcemide. The postmitoehondrial supernatant obtained from these cells was then partitioned b y sucrose gradient centrifugation into four fractions (Fig. 2a), and each fraction was assayed for the snRNA's. The results are shown in Figs. 2b-2e and summarized in Fig. 2f. It will be noted that each of the principal snRNA's was found in all four fractions; they thus sediment quite heterogeneously. Each of the species analyzed, furthermore, has a different distribution; species A, which appears to be associated with the nucleolus in interphase nuclei8, has a very broad, possibly bimodal distribution in mitotic cells; species C is found mainly in structures sedimenting at less than 60-S; and species D is found largely in the same size range as ribosomes and small polyribosomes. These results suggest that ill mitotic cells the snRNA's are contained in particles which are heterogeneous in size, but that the particles containing the different RNA's have different size spectra.
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The snRNA's in mitotic cells clearly sediment much faster than naked RNA. It is known, however, that ribosomal RNA added to a cytoplasmic extract at low ionic strength sediments more rapidly than ribosomal RNA alone because of its tendency to associate with a variety of cytoplasmic proteins xz. It therefore seemed Biochim. Biophys. Acta, 232 (i971) 3o6-313
3Io
A. REIN
possible that the snRNA--containing "particles" observed in mitotic cells were actually random aggregates of cellular proteins and snRNA's. To test this possibility, a reconstruction experiment was performed. An exponentially growing culture was labeled overnight with ~H!uridine , and nuclear RNA was prepared from it by phenol extraction. A second, unlabeled culture was synchronized and harvested after 82 0/o of the cells had been arrested in mitosis with colcemide. The cells were washed with Earle's saline and then resuspended in RSB buffer containing the 3H-labeled nuclear RNA. This suspension was homogenized and fractionated into the "nuclear" pellet, the mitochondrial pellet, and four fractions taken from a sucrose gradient as described in Fig. 2. Each fraction was then analyzed for the snRNA's. The results were tabulated only for species D (Fig. 3) but were essentially the same for species A, C, G', IOO
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Fig. 3. Distribution of *H-labeled nuclear R N A after mixing with unlabeled mitotic ceils. 5 " lOS cells were grown for 18 h in 25 ml of m e d i u m containing 5 ° .uC of [SH]uridine. Nuclear R N A was t h e n isolated b y phenol extraction. 3 • lOT cells in IOO ml of m e d i u m were treated with 2 mM t h y m i d i n e for I5 h and t h e n centrifuged and resuspended in ioo ml of fresh medium. 2 mM t h y m i d i n e was added 9 h later. The cells were again centrifuged and resuspended in 17o ml of fresh m e d i u m after 12 h in thymidine. Colcemide (o.6/~g/ml) was added 6 h later. After 8 h in colcemide, w h e n the mitotic index was 82 %, the cells were centrifuged, washed in Earle's saline, and incubated for io min in RSB buffer containing the SH-labeled nuclear RNA. T h e y were t h e n broken w i t h a Dounce homogenizer. A " n u c l e a r " pellet was first obtained b y centrifuging the h o m o g e n a t e for 2 min at IOOO× g ; a mitochondrial pellet was t h e n obtained b y centrifuging the resulting s u p e r n a t a n t at 12 ooo × g for IO rain. The p o s t m i t o c h o n d r i a l s u p e r n a t a n t was layered on a 15-3o % (w/w) sucrose gradient in R S B buffer and centrifuged for 6 h at 25 ooo r e v . / m i u in the Spinco SW 25.3 rotor. R N A ' s were extracted from the pellets and three regions of the gradient, and electrophoresed for 6. 5 h. The figure shows the percentage of the 8H-labeled species D found in each cell fraction.
and H as well. I t was found that the snRNA's had some tendency to adhere to chromosomes and mitochondria, but the bulk of them appeared at the top of the gradient. Most strikingly, the pellet and bottom two-thirds of the gradient, which contained a major fraction of the snRNA's in mitotic cells (Fig. 2), each contained less t h a n 2 °/o of the snRNA's in the reconstruction experiment. The distributions seen in the mitotic cells thus do not seem to be the result of aggregation of free snRNA with cellular proteins. It would be of great interest to know whether the snRNA-containing particles observed in mitotic cells also exist within interphase nuclei. WEINBERG AND PENMAN8 Biochim. Biophys. Acta, 232 (i97 I) 3o6-313
SMALL NUCLEAR
R N A IN MITOTIC CELLS
311
found that when snRNA's are extracted from isolated nuclei by raising the salt concentration, they are in structures which sediment at less than IO S. It is possible, however, that snRNA's are contained in the same particles in mitotic and interphase cells, but that the salt concentrations which release the snRNA's from the nucleus also disrupt the particles. To test this hypothesis, the salt lability of the particles from mitotic cells was examined in the following experiment. The postmitochondrial supernatant of a prelabeled, mitotic culture was prepared as in Fig. 2. It was then made o.3 M in NaC1 and 0.03 M in MgC12, since this is the solution used s to quantitatively elute the nucleoplasmic snRNA's from nuclei; and centrifuged in a sucrose gradient containing these salts. Fractions were taken from the gradient (Fig. 4 a) and analyzed for snRNA's. As will be seen (Figs. 4b-4f ), the increased ionic strength shifted the snRNA's almost entirely into the 0-30 S region of the gradient. (The sedimentation rate of ribosomes and small ribosomal subunits is also reduced somewhat in these salt concentrations.) Thus, the structures containing the snRNA's in mitotic cells are quite labile to moderate salt concentrations; the results of W E I N B E R G AND PENMAN are therefore consistent with the idea that these structures also exist in interphase nuclei.
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Fig. 4. Size of particles from mitotic cells containing s n R N A ' s in 60 % H S B buffer. The other half of the postmitochondrial supernatant used in Fig. 2 w a s m a d e 0. 3 M NaC1 and o.o 3 M MgCI~ and layered on a 15-3o °/ 5 (w/w) sucrose gradient containing these salts and o.oi M Tris, p H 7.4It w a s centrifuged for 6 h at 25 ooo rev./min in the Spinco S W 25. 3 rotor. Fractions were collected and analyzed as described in Fig. 2. (a) Absorbance profile of the gradient. (b) Pellet of the gradient. (c) Region I of the gradient. (d) Region II of the gradient. (e) Region I I I of the gradient. (f) Distribution of each R N A species in different regions of the gradimlt.
DISCUSSION
It has been shown here that when mitotic cells are fractionated at low ionic strength, the snRNA's sediment as if they were contained in particles which are heterogeneous in size but predominantly less than 18o S. Each of the snRNA's analyzed was distributed differently over this size i ange. Biochim. Biophys. Acta, 232 (i97 I) 3 o 6 - 3 1 3
312
A. REIN
Do these particles actually exist within the cell, or are they artifacts of cell fractionation? A reconstruction experiment showed that when naked snRNA's are mixed with mitotic cells they sediment with a completely different distribution from those mentioned above. Thus the present results do not seem to represent the aggregation of free RNA in mitotic cells with other cellular constituents. The converse possibility, that the particles observed are actually breakdown products of larger structures, is more difficult to exclude, particularly in view of their heterogeneity. Colcemide was used to collect mitotic cells in all of these experiments; although this agent does not remove the snRNA's from interphase nuclei, it doubtless disrupts large microtubule-containing structures in mitotic cells. This could conceivably cause the formation of the particles described in this study. Larger structures might also be degraded during cell fractionation. This cannot be ruled out but seems unlikely, since all steps were performed at o ° in solutions of low ionic strength, conditions which tend to preserve the electrostatic bonds common in nucleoproteins. Verylittle DNA appeared in the postmitochondrial supernatant, and no degradation of ribosomes was observed. Assuming that the particles detected in the present experiments actually are the form in which the snRNA's pass through mitosis, it would be of interest to learn more about their structure. It seems extremely unlikely that a single snRNA molecule could organize enough proteiu around it to form particles this large (for example, a ribonucleoprotein particle with the molecular weight of a ribosome but containing only one snRNA molecule would be more than 98 °/o protein). Thus the particles might include other macromolecules as well, or might be "packages" containing many snRNA molecules per particle. We may also consider the possibility that these particles exist within interphase nuclei as well as in mitotic cells, that is, that they do not change when they are reincorporated into the daughter nucleus after each cell division. It is known that the snRNA's are rather loosely bound to the interphase chromatin, and when eluted by moderate salt concentrations sediment quite slowly8. However, it has also been found 12 that the snRNA's adhere to bentonite even when extracted from interphase nuclei in 0.8 M NaC1 plus 0.08 M MgCI~. This indicates that some other material, possibly protein, remains bound to the snRNA's at high ionic strength, but not enough to cause them to sediment rapidly. It is the bonds between these "core particles" and the rest of the nucleus which are so salt labile. These may be the salt-labile bonds detected in the present study of the mitotic particles.
ACKNOWLEDGEMENTS
This work was performed under the tenure of the American Cancer Society Postdoctoral Fellowship PF-36oA, and was also supported by grant CA-o8416-o 5 of the National Institutes of Health and Grant GB-8515 of the National Science Foundation awarded to Prof. Sheldon Penman. The author wishes to acknowledge the excellent advice he received from Dr. Penman throughout the course of this work, and also Mr. Hung Fan's expert guidance in cell synchrony and the skillful technical assistance of Mrs. Deana Fowler and Miss Linda Colwell. Biochim. Biophys. Acta, 232 (1971) 3o6-313
SMALL NUCLEAR
RNA IN MITOTIC CELLS
313
REFERENCES i 2 3 4 5 6 7 8 9 io ii 12
J. E. DARNELL, JR., Bacteriol. Rev., 32 (1968) 262. T. NAKAMURA, A. V~. PRESTAYKO AND H. BUSCH, J. Biol. Chem., 243 (1968) 1368. R. A. WEINBERG AND S. PENMAN, J. Mol. Biol., 38 (1968) 289. C. J. LARSEN, b'. GALIBERT, A. HAMPE AND M. BOIRON, Compt. Rend., 267 (1968) I I . W . V. ZAPISEK, A. G. SAPONARA AND M. D. ENGER, Biochemistry, 8 (1969) 117o. A. REIN AND S. PENMAN, Biochim. Biophys. Acta, 19o (1969) I. C. V~r. DINGMAN AND A. C. PEACOCK, Biochemistry, 7 (1968) 659. R. A. WEINBERG AND S. PENMAN, Biochim. Biophys. Acta, 19o (1969) io. S. PENMAN, J. Mol. Biol., 17 (1966) 117. J. J. PENE, E. KI~IG~IT, JR. AND ]. E. DARNELL, JR., J. Mol. Biol., 33 (1968) 609. D. BALTIMORE AND A, S. HUANG, J. ]~lol. Biol., 47 (197o) 263. R. A. WALTERS, L. R. GURLEY, A. G. SAI~ONARAAND lV[. D. ENGER, Biochim. Biophys. Acta, 199 (197 ° ) 525 .
Biochim. Biophys. Acta, 232 (I97 I) 3o6-313
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96780
T H E SMALL MOLECULAR W E I G H T M O N O D I S P E R S E N U C L E A R RNA's IN MITOTIC CELLS
ALAN R E I N Bionetics Research Laboratories, Inc., 7300 Pearl St., Bethesda, Md. 2ooi 4 (U.S.A.)
(Received September 7th, 197o )
SUMMARY
I. Since the small molecular weight monodisperse nuclear RNA's (snRNA's) are permanently associated with the nuclei of growing cells, they must be re-incorporated into the daughter nuclei after each cell division. Their state was investigated in H e L a cells arrested in metaphase with colcemide. When cells were fractionated at low ionic strength, the snRNA's were found to be in structures sedimenting over a wide range, but principally between 30 and 18o S. Different snRNA's were distributed differently over this size range. 2. A reconstruction experiment indicated that this sedimentation distribution was not the result of aggregation of free snRNA's with other celhllar materials during cell fractionation. 3- The structures containing the snRNA's in mitotic cells sediment much more slowly in gradients containing 0.3 M NaC1 plus 0.03 M MgC12 than in the low ionic strength medium mentioned above. 4. The results suggest that the snRNA's are carried in relatively small particles during mitosis. These particles m a y also exist in interphase nuclei; their salt lability is compatible with this possibility.
INTRODUCTION
It has recently been discovered that the nuclei of vertebrate cells contain, in addition to the ribosomal precursor RNA's and the nuclear heterodisperse RNA ~, a third class of RNA species. This class consists of nine RNA species, each of discrete molecular weight, ranging from 3 " 104 to 8 • lO 4. They are extensively methylated and metabolically quite stable. They are found only in the cell nucleus. They have now been reported in a variety of tissue culture lines 2-6 and in embryonic 3 and adult 7 tissues. Their functions are completely unknown. They will be referred to here as the "small molecular weight monodisperse nuclear R N A ' s " (snRNA's). When an exponentially growing cell population is labeled briefly with an RNA precursor and then grown in nonradioactive medium, the labeled snRNA's are found to remain associated with the nuclei over several cell generations 3. They must, therefore, re-associate with the nucleus after each division. This re-association Abbreviation: snRNA, small molecular weight monodisperse nuclear RNA. I3iochim. Biophys. Acta, 232 (I97 I) 3o6-313
SMALL NUCLEAR
RNA
IX MITOTIC CELLS
307
has in fact been demonstrated directly in synchronized cultures 8. In an a t t e m p t to understand the mechanism of this specific association with the nucleus after mitosis, we were prompted to investigate the state of these RNA's during metaphase. In particular, one possibility was that the RNA's are directed into the daughter nuclei b y being attached to the chromosomes. The results to be presented indicate that, rather, the snRNA's are contained in much smaller particles in mitotic cells. MATERIALS AND METHODS
Buffers used included o.oi M NaCl-o.ooI5 M MgC12-o.oI M Tris buffer, p H 7.4 (RSB buffer); 0. 5 M NaCl-o.o 5 M MgCl~-o.oI M Tris buffer, p H 7.4 (HSB buffer); and o.I M NaCl-o.ooI M EDTA-o.5 ~o sodium dodecyl sulfate-o.oI M "Iris buffer, p H 7.4 (SDS buffer). [5-zHlUridine (25 C/mmole) was purchased from New England Nuclear Corp. Thymidine was purchased from Calbiochem. All experiments were done with spinner cultures of H e L a type S 3 cells. The procedures for growing and synchronizing these cells, and for extraction and analysis of the snRNA's, were as described b y WEINBERG AND PENMAN3,8. Briefly, cells are grown in Joklik-modified Eagle's medium at a concentration of 2 • lO5-4 • lO 5 cells/ mh Populations of metaphase cells are obtained by synchronizing the cells b y double thymidine block and then collecting the cells in mitosis in colcemide. Cells were fractionated 9 at o °. RNA's were extracted with phenol and chloroform ~in the presence of sodium dodecyl sulfate and EDTA. Extractions were performed at room temperature, since at higher temperatures the 28-S associated RNA 1° is liberated from 28-S ribosomal RNA and interferes with the electrophoretic analysis of snRNA's in mitosis. Control experiments have shown that extractions at room temperature and at 55 ° yield the same amount of snRNA's from interphase cells. All cell fractions were treated with DNase after phenol extraction 3, and then electrophoresed on IO °/o polyacrylamide gels at 7 V/cm. The gels were then cut into I-ram slices and each slice was digested with N H 4 0 H and counted in a liquid scintillation counter. All gels contained 14C-labeled marker RNA as well as the 3H-labeled RNA being analyzed. These markers were an important aid in identifying the individual RNA peaks with confidence; however, for simplicity, they have been omitted from all but one of the figures. Although nine species of snRNA's, namely A, B, C, D, F, G', H, K, and L, have been described in H e L a cells8, only A, C, and D will be studied here. The other species are either present in too small amounts, or have electrophoretic mobilities too close to the cytoplasmic species (i.e. 5-S ribosomal RNA and transfer RNA) to allow analysis in mitotic cells. The partition of cellular DNA into the different cell fractions was analyzed b y measuring the radioactivity which remained trichloroacetic acid precipitable after dilution in SDS buffer and incubation overnight at 37 ° in 0.3 M NaOH. In long labeling periods such as those employed in these experiments, cells which are synthesizing DNA convert a considerable fraction of the [5-3H]uridine label into Es-SH]deoxycytidine and incorporate it into DNA. Mitotic indices were determined under the phase contrast microscope. The cells were first centrifuged and resuspended in RSB buffer to increase the visibility of the nucleus in nonmitotic cells. Biochim. Biophys. Acta, 232 (1971) 3o6-313
308
A. REIN
RESULTS
The first experiment to be presented is a single centrifugation fractionation oi prelabeled mitotic cells blocked in metaphase with colcemide. A culture of HeLa cells was labeled with [3H]uridine between the first and second thymidine blocks. As is shown in Fig. Ia, the culture was divided into two parts upon release from the second thymidine block. One was treated with colcemide for the first 7 h after release, and then harvested; none of the cells in this portion had yet entered mitosis. The other was treated with colcemide from 7 to 14. 5 h after release, so that when it was harvested 80 % of the cells were in metaphase. The two suspensions were homogenized and fractionated by differential centrifugation, and each fraction was analyzed for DNA and the snRNA's (Figs. Ib-If). In the interphase culture, the nuclear pellet contained nearlv, all the snRNA's and 93 O/;oof the DNA. The control shows that colcemide treatment per se does not liberate snRNA's from large nucleoprotein structures. In the mitotic culture, however, the snRNA's were found mainly in the postmitochondrial supernatant, which contained only 5 % of the DNA. (Although 20 % of the cells had visible nuclear membranes, the "nuclear" pellet with 7 ° % of the DNA, contained no detectable snRNA. It seems likely that the snRNA's in the mitochondrial pellet were derived from the remaining interphase cells. These cells were evidently not proceeding into mitosis (Fig. Ia) and perhaps had particularly fragile nuclei. Comparison of the yields of the snRNA's from the two cultures shows that the RNA's were completely recovered from the mitotic cells. l~er~b~ CelN:C¥ 0 q l 0 s m
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a Fig'. i. s n R N A ' s in d i f f e r e n t cell f r a c t i o n s of i n t e r p h a s e a n d m i t o t i c cells. 2 • lO7 cells in i o o ml of m e d i u m were t r e a t e d w i t h 2 mM t h y m i d i n e for 15 h a n d t h e n c e n t r i f u g e d a n d r e s u s p e n d e d in i o o ml of fresh m e d i u m . 15o/zC of t S H l u r i d i n e were a d d e d 6. 5 h later, a n d 2 mlV[ t h y m i d i n e w a s a d d e d 2 h a f t e r t h e label. The cells were a g a i n c e n t r i f u g e d a n d r e s u s p e n d e d in IOO ml of fresh m e d i u m a f t e r 14 h in t h y m i d i n e . Colcemide (0.6/~g/ml} w a s a d d e d t o o n e - t h i r d of t h e c u l t u r e u p o n r e s u s p e n s i o n in fresh m e d i u m . 7 h l a t e r t h e s e cells were h a r v e s t e d a n d c o l c e m i d e a d d e d to t h e r e m a i n d e r of t h e culture. The h a r v e s t e d cells were r i n s e d in E a r l e ' s saline, i n c u b a t e d for i o rain a t o ° in R S B buffer a n d b r o k e n w i t h a D o u n c e h o m o g e n i z e r . N u c l e i w e re p e l l e t e d b y cent r i f u g a t i o n a t i o o o × g for 3 rain a n d d i g e s t e d b y d e o x y r i b o n u c l e a s e in H S B buffer. 7.5 h l a t e r t h e r e m a i n d e r of t h e c u l t u r e w a s f r a c t i o n a t e d b y e x a c t l y t h e s a m e proc e dure , e x c e p t t h a t a second c e n t r i f u g a t i o n of 12 ooo × g for i o rain was p e r f o r m e d on t h e s u p e r n a t a n t of t h e fi rs t c e n t r i f u g a tion. A l i q u o t s of all cell f r a c t i o n s were r e m o v e d for d e t e r m i n a t i o n of r a d i o a c t i v i t y i n D N A , a n d RBTA's were e x t r a c t e d a n d e l e c t r o p h o r e s e d for 8 h. (a) M i t o t i c indices of t h e c o l c e m i d e - c o n t a i n i n g c u l t u r e s . (b) N u c l e a r p e l l e t of t h e i n t e r p h a s e cells. (c) C y t o p l a s m of t h e i n t e r p h a s e cells. (d) " N u c l e a r " p e l l e t of t h e m i t o t i c cells. (e) 12 ooo × g p e l l e t of t h e m i t o t i c cells (f) 12 ooo × g supern a t a n t of t h e m i t o t i c cells. u
Biochim. Biophys. Acta, 232 (I971) 3o6-313
SMALL NUCLEAR
RNA
309
IN MITOTIC CELLS
An experiment was then performed to determine the size of the structures containing the snRNA's in mitotic cells. A culture was labeled with [3H~uridine during synchronization and the cells were collected in mitosis with colcemide. The postmitoehondrial supernatant obtained from these cells was then partitioned b y sucrose gradient centrifugation into four fractions (Fig. 2a), and each fraction was assayed for the snRNA's. The results are shown in Figs. 2b-2e and summarized in Fig. 2f. It will be noted that each of the principal snRNA's was found in all four fractions; they thus sediment quite heterogeneously. Each of the species analyzed, furthermore, has a different distribution; species A, which appears to be associated with the nucleolus in interphase nuclei8, has a very broad, possibly bimodal distribution in mitotic cells; species C is found mainly in structures sedimenting at less than 60-S; and species D is found largely in the same size range as ribosomes and small polyribosomes. These results suggest that ill mitotic cells the snRNA's are contained in particles which are heterogeneous in size, but that the particles containing the different RNA's have different size spectra.
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The snRNA's in mitotic cells clearly sediment much faster than naked RNA. It is known, however, that ribosomal RNA added to a cytoplasmic extract at low ionic strength sediments more rapidly than ribosomal RNA alone because of its tendency to associate with a variety of cytoplasmic proteins xz. It therefore seemed Biochim. Biophys. Acta, 232 (i971) 3o6-313
3Io
A. REIN
possible that the snRNA--containing "particles" observed in mitotic cells were actually random aggregates of cellular proteins and snRNA's. To test this possibility, a reconstruction experiment was performed. An exponentially growing culture was labeled overnight with ~H!uridine , and nuclear RNA was prepared from it by phenol extraction. A second, unlabeled culture was synchronized and harvested after 82 0/o of the cells had been arrested in mitosis with colcemide. The cells were washed with Earle's saline and then resuspended in RSB buffer containing the 3H-labeled nuclear RNA. This suspension was homogenized and fractionated into the "nuclear" pellet, the mitochondrial pellet, and four fractions taken from a sucrose gradient as described in Fig. 2. Each fraction was then analyzed for the snRNA's. The results were tabulated only for species D (Fig. 3) but were essentially the same for species A, C, G', IOO
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Fig. 3. Distribution of *H-labeled nuclear R N A after mixing with unlabeled mitotic ceils. 5 " lOS cells were grown for 18 h in 25 ml of m e d i u m containing 5 ° .uC of [SH]uridine. Nuclear R N A was t h e n isolated b y phenol extraction. 3 • lOT cells in IOO ml of m e d i u m were treated with 2 mM t h y m i d i n e for I5 h and t h e n centrifuged and resuspended in ioo ml of fresh medium. 2 mM t h y m i d i n e was added 9 h later. The cells were again centrifuged and resuspended in 17o ml of fresh m e d i u m after 12 h in thymidine. Colcemide (o.6/~g/ml) was added 6 h later. After 8 h in colcemide, w h e n the mitotic index was 82 %, the cells were centrifuged, washed in Earle's saline, and incubated for io min in RSB buffer containing the SH-labeled nuclear RNA. T h e y were t h e n broken w i t h a Dounce homogenizer. A " n u c l e a r " pellet was first obtained b y centrifuging the h o m o g e n a t e for 2 min at IOOO× g ; a mitochondrial pellet was t h e n obtained b y centrifuging the resulting s u p e r n a t a n t at 12 ooo × g for IO rain. The p o s t m i t o c h o n d r i a l s u p e r n a t a n t was layered on a 15-3o % (w/w) sucrose gradient in R S B buffer and centrifuged for 6 h at 25 ooo r e v . / m i u in the Spinco SW 25.3 rotor. R N A ' s were extracted from the pellets and three regions of the gradient, and electrophoresed for 6. 5 h. The figure shows the percentage of the 8H-labeled species D found in each cell fraction.
and H as well. I t was found that the snRNA's had some tendency to adhere to chromosomes and mitochondria, but the bulk of them appeared at the top of the gradient. Most strikingly, the pellet and bottom two-thirds of the gradient, which contained a major fraction of the snRNA's in mitotic cells (Fig. 2), each contained less t h a n 2 °/o of the snRNA's in the reconstruction experiment. The distributions seen in the mitotic cells thus do not seem to be the result of aggregation of free snRNA with cellular proteins. It would be of great interest to know whether the snRNA-containing particles observed in mitotic cells also exist within interphase nuclei. WEINBERG AND PENMAN8 Biochim. Biophys. Acta, 232 (i97 I) 3o6-313
SMALL NUCLEAR
R N A IN MITOTIC CELLS
311
found that when snRNA's are extracted from isolated nuclei by raising the salt concentration, they are in structures which sediment at less than IO S. It is possible, however, that snRNA's are contained in the same particles in mitotic and interphase cells, but that the salt concentrations which release the snRNA's from the nucleus also disrupt the particles. To test this hypothesis, the salt lability of the particles from mitotic cells was examined in the following experiment. The postmitochondrial supernatant of a prelabeled, mitotic culture was prepared as in Fig. 2. It was then made o.3 M in NaC1 and 0.03 M in MgC12, since this is the solution used s to quantitatively elute the nucleoplasmic snRNA's from nuclei; and centrifuged in a sucrose gradient containing these salts. Fractions were taken from the gradient (Fig. 4 a) and analyzed for snRNA's. As will be seen (Figs. 4b-4f ), the increased ionic strength shifted the snRNA's almost entirely into the 0-30 S region of the gradient. (The sedimentation rate of ribosomes and small ribosomal subunits is also reduced somewhat in these salt concentrations.) Thus, the structures containing the snRNA's in mitotic cells are quite labile to moderate salt concentrations; the results of W E I N B E R G AND PENMAN are therefore consistent with the idea that these structures also exist in interphase nuclei.
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Fig. 4. Size of particles from mitotic cells containing s n R N A ' s in 60 % H S B buffer. The other half of the postmitochondrial supernatant used in Fig. 2 w a s m a d e 0. 3 M NaC1 and o.o 3 M MgCI~ and layered on a 15-3o °/ 5 (w/w) sucrose gradient containing these salts and o.oi M Tris, p H 7.4It w a s centrifuged for 6 h at 25 ooo rev./min in the Spinco S W 25. 3 rotor. Fractions were collected and analyzed as described in Fig. 2. (a) Absorbance profile of the gradient. (b) Pellet of the gradient. (c) Region I of the gradient. (d) Region II of the gradient. (e) Region I I I of the gradient. (f) Distribution of each R N A species in different regions of the gradimlt.
DISCUSSION
It has been shown here that when mitotic cells are fractionated at low ionic strength, the snRNA's sediment as if they were contained in particles which are heterogeneous in size but predominantly less than 18o S. Each of the snRNA's analyzed was distributed differently over this size i ange. Biochim. Biophys. Acta, 232 (i97 I) 3 o 6 - 3 1 3
312
A. REIN
Do these particles actually exist within the cell, or are they artifacts of cell fractionation? A reconstruction experiment showed that when naked snRNA's are mixed with mitotic cells they sediment with a completely different distribution from those mentioned above. Thus the present results do not seem to represent the aggregation of free RNA in mitotic cells with other cellular constituents. The converse possibility, that the particles observed are actually breakdown products of larger structures, is more difficult to exclude, particularly in view of their heterogeneity. Colcemide was used to collect mitotic cells in all of these experiments; although this agent does not remove the snRNA's from interphase nuclei, it doubtless disrupts large microtubule-containing structures in mitotic cells. This could conceivably cause the formation of the particles described in this study. Larger structures might also be degraded during cell fractionation. This cannot be ruled out but seems unlikely, since all steps were performed at o ° in solutions of low ionic strength, conditions which tend to preserve the electrostatic bonds common in nucleoproteins. Verylittle DNA appeared in the postmitochondrial supernatant, and no degradation of ribosomes was observed. Assuming that the particles detected in the present experiments actually are the form in which the snRNA's pass through mitosis, it would be of interest to learn more about their structure. It seems extremely unlikely that a single snRNA molecule could organize enough proteiu around it to form particles this large (for example, a ribonucleoprotein particle with the molecular weight of a ribosome but containing only one snRNA molecule would be more than 98 °/o protein). Thus the particles might include other macromolecules as well, or might be "packages" containing many snRNA molecules per particle. We may also consider the possibility that these particles exist within interphase nuclei as well as in mitotic cells, that is, that they do not change when they are reincorporated into the daughter nucleus after each cell division. It is known that the snRNA's are rather loosely bound to the interphase chromatin, and when eluted by moderate salt concentrations sediment quite slowly8. However, it has also been found 12 that the snRNA's adhere to bentonite even when extracted from interphase nuclei in 0.8 M NaC1 plus 0.08 M MgCI~. This indicates that some other material, possibly protein, remains bound to the snRNA's at high ionic strength, but not enough to cause them to sediment rapidly. It is the bonds between these "core particles" and the rest of the nucleus which are so salt labile. These may be the salt-labile bonds detected in the present study of the mitotic particles.
ACKNOWLEDGEMENTS
This work was performed under the tenure of the American Cancer Society Postdoctoral Fellowship PF-36oA, and was also supported by grant CA-o8416-o 5 of the National Institutes of Health and Grant GB-8515 of the National Science Foundation awarded to Prof. Sheldon Penman. The author wishes to acknowledge the excellent advice he received from Dr. Penman throughout the course of this work, and also Mr. Hung Fan's expert guidance in cell synchrony and the skillful technical assistance of Mrs. Deana Fowler and Miss Linda Colwell. Biochim. Biophys. Acta, 232 (1971) 3o6-313
SMALL NUCLEAR
RNA IN MITOTIC CELLS
313
REFERENCES i 2 3 4 5 6 7 8 9 io ii 12
J. E. DARNELL, JR., Bacteriol. Rev., 32 (1968) 262. T. NAKAMURA, A. V~. PRESTAYKO AND H. BUSCH, J. Biol. Chem., 243 (1968) 1368. R. A. WEINBERG AND S. PENMAN, J. Mol. Biol., 38 (1968) 289. C. J. LARSEN, b'. GALIBERT, A. HAMPE AND M. BOIRON, Compt. Rend., 267 (1968) I I . W . V. ZAPISEK, A. G. SAPONARA AND M. D. ENGER, Biochemistry, 8 (1969) 117o. A. REIN AND S. PENMAN, Biochim. Biophys. Acta, 19o (1969) I. C. V~r. DINGMAN AND A. C. PEACOCK, Biochemistry, 7 (1968) 659. R. A. WEINBERG AND S. PENMAN, Biochim. Biophys. Acta, 19o (1969) io. S. PENMAN, J. Mol. Biol., 17 (1966) 117. J. J. PENE, E. KI~IG~IT, JR. AND ]. E. DARNELL, JR., J. Mol. Biol., 33 (1968) 609. D. BALTIMORE AND A, S. HUANG, J. ]~lol. Biol., 47 (197o) 263. R. A. WALTERS, L. R. GURLEY, A. G. SAI~ONARAAND lV[. D. ENGER, Biochim. Biophys. Acta, 199 (197 ° ) 525 .
Biochim. Biophys. Acta, 232 (I97 I) 3o6-313