ANALYTICAL
BIOCHEMISTRY
Isolation
66, 18-28 (1975)
of Messenger
by Chromatography R.E.
RNA
from
Polysomes
on Oligo(dT)-Cellulose
PEMBERTON, P. LIBERTI, AND C. BACLIONI
Department Cambridge,
of Biology, Massachusetts Institute of Technology, Massachusetts, and Laboratory of Cell Biology, via Romagnosi 18/A, Rome, Italy
Received April 15, 1974; accepted December 24, 1974 A method for the isolation of messenger RNA (mRNA) from polysomes is described. Pofysomes are dissolved in a solution containing0.5 M NaCl and Na dodecyl sulphate and applied to an oligo(dT)-cellulose column. RNA speciescontaining poly(A) sequences are retained by the column, whereas ribosomal proteins and other RNA species are washed off. The column is then eluted with a buffer not containing NaCI. mRNA from HeLa cells and from duck reticulocytes has been fractionated in this way. When fractionated on sucrose gradients, 10 s globin mRNA is obtained in addition to a 20 s component, which can be translated in a cell free system into duck globin. This 20 s RNA is an aggregate of mRNA, which can be disaggregated. Experiments with HeLa cells have shown that the only mRNA species which is not retained by oligo(dT)-cellulose is histone mRNA; this mRNA does not contain a poly(A) sequence.
Rabbit globin mRNA has been isolated by Aviv and Leder (1) from rabbit reticulocyte RNA by chromatography on oligo(dT)-cellulose. Subsequently, mRNA for the light chain of a mouse immunoglobulin has been isolated by the same method from the RNA of a myeloma and its identity established by translation in an ascites cell-free system (2). This chromatographic method can thus be generally used for the isolation of mRNA’s containing poly(A) sequences. So far, phenol extraction has been used for the preliminary extraction of RNA from polysomes (1,2). Phenol extraction presents some disadvantages, particularly the risk of losing part or all of the poly(A) sequence (3). This can be avoided by using extraction procedures that have been shown to preserve the integrity of poly(A) (3). It would be preferable, however, to avoid any extraction and to isolate mRNA by mild procedures. We have thus isolated mRNA by direct chromatography of resuspended polysomes on oligo(dT)-cellulose. The polysomes are dissociated by the use of the strong detergent Na dodecyl sulphate which is present throughout the chromatography. The RNA isolated has been analyzed by sucrose gradients and aggregation of duck globin mRNA into a discrete 20 s component has been noticed. The formation of this 18 Copyright Al, &“htr
@ 1975 by Academic d*a..-^rl ..^. :.- ‘-
Press, Inc. -
*
ISOLATION
aggregate and the behavior of histone mRNA, poly(A) sequences (4), have also been studied. MATERIALS
19
OF mRNA
AND
which does not contain
METHODS
Column chromatography on oligo(dT)-cellulose. A 0.5 X 4-cm cdumn of oligo(dT)-cellulose, supplied by Collaborative Research Inc., was equilibrated with 0.5 M NaCl, 0.5% Na dodecyl sulphate and 0.01 M Tris buffer, pH 7.5 (starting buffer). The samples applied to the column were dissolved in water and then diluted with an equal volume of twice-concentrated starting buffer. The column was washed with approximately 30 ml of starting buffer and then eluted with 0.5% Na dodecyl sulphate, 0.01 M Tris buffer, pH 7.5. All the operations, after the addition of starting buffer to the samples, were carried out at room temperature. RNA was precipitated with two volumes of ethanol at 20°C after adjusting the NaCl concentration to 0.1 M. Polysomes. HeLa cells were grown in minimal Eagle’s medium (5) in spinner bottles. The cells were collected by centrifugation and resuspended at 2 X 10 cell/ml. The cells were incubated for 30 min with 40 rig/ml of actinomycin D to inhibit the synthesis of ribosomal RNA (6) and then for 2 hr with 40 &i/ml of [5-3H]uridine (56.7 Cilmole; New England Nuclear). The cells were collected by centrifugation, washed with ice-cold Earle’s saline and resuspended in RSB buffer (10 mM NaCl, 10 mM Tris, pH 7.4, and 1.5 mM MgCl,; I ml of buffer/lo7 cells). After 10 min the cells were homogenized and the postmitochondrial supernatant obtained by centrifugation for IO min at 20,000 g. This was applied over 30% sucrose in RSB and the polysomes pelletted by a 3-hr centrifugation at 150,OOOg. Duck reticulocytes were obtained as previously described (7) and reticulocyte polysomes prepared as described above. Preparation and analysis of RNA. Polysome pellets were dissolved in SDS buffer (0.1 M NaCl, 10 mM Tris pH 7.4, 10 mM ethylene diamino tetraacetic acid and 0.5% Na dodecyl sulphate) to extract RNA as previously described (8). The polysome pellets were dissolved in starting buffer (see above) for fractionation of the RNA by oligo(dT)-cellulose chromatography. The RNA obtained by either method was precipitated with ethanol and further characterized by sucrose gradient centrifugation or acrylamide-gel electrophoresis. Centrifugations were run as described in the legends to figures, using 5-20% sucrose gradients in SDS buffer. Fractions were counted after precipitation with 5% trichloroacetic acid. For the electrophoretic analysis 3% acrylamide gels were run for 150 min, sliced and counted as previously described (9,lO). RNA fractions were assayed in the ascites cell free system described in detail previously (11).
20
PEMBERTON,
LIBERTI
AND BAGLIONI
Preparation and analysis of proteins. HeLa cell histones uniformly labeled with [14C]lysine were prepared as described previously (13). Duck hemoglobin uniformly labeled with [35S]methionine was prepared by incubating 1 ml of duck reticulocytes for 18 hr with 100 #Zi of [35S]methionine (90 Cilmole; New England Nuclear) as previously described (12); the cells were lysed and the postmitochondrial supematant fraction prepared as described above. This protein and the product of the cell-free system were analyzed by electrophoresis on 10% acrylamide gels. These were prepared, run, fractionated and counted as previously described ( 13).
RESULTS Fractionation
of HeLa Cells mRNA
In order to fractionate RNA on oligo(dT)-cellulose columns without prior phenol extraction, we have used buffers containing the detergent Na dodecyl sulphate to dissolve polysomes and have included the same detergent in the buffers used to equilibrate and elute the columns. It was thus necessary to substitute the KC1 used previously (1,2) with NaCl to avoid precipitation of the detergent. In a preliminary experiment we established that even very basic proteins, like histones, are not absorbed by oligo(dT)-cellulose. In this experiment we mixed 0.03 mg of HeLa cells RNA labeled with C3H]uridine in the presence of actinomycin D and extracted from polysomes (see Methods) with 0.01 mg of HeLa cell histones labeled with [r4C]lysine; 1 mg of yeast tRNA was added as carrier to allow us to follow the AzeO pattern. In the chromatography on oligo(dT)-cellulose (not shown), 98% of the histones applied were recov-
FRACTIONS
FIG. 1. Chromatography of HeLa cell RNA on oligo(dT)-cellulose. (A), Elution pattern of the RNA labeled with [3H]uridine in presence of 40 rig/ml of actinomycin D (see Methods). The arrow denotes the start of the low salt elution. Two-milliliter fractions were collected; (B), analysis by centrifugation on sucrose gradients of the RNA eluted in peaks 1 and 2 of A. The patterns obtained in two separate gradients are superimposed and the position of marker RNA species run on parallel gradients is shown for reference. The gradients were centrifuged for 14 hr at 25,000 rpm.
ISOLATION
OF mRNA
21
ered in peak 1 (see Fig. I), and nearly 100% of the HeLa RNA was recovered, 44% in peak 1 and 66% in peak 2 (the fraction eluted with buffer not containing NaCl; see Methods and Fig. 1A). Polysomes prepared from HeLa ceils labeled with [3H]uridine were dissolved in starting buffer and fractionated by chromatography on oligo(dT)-cellulose (Fig. 1A). Again, the RNA was found partitioned, 38% in peak 1 and 62% in peak 2. The fractions corresponding to each peak were pooled and the RNA precipitated and analyzed by sucrose gradient centrifugation (Fig. 1B). The RNA of peak 1 consisted mainly of tRNA (4 s RNA), whereas the RNA of peak 2 sedimented in a rather broad peak, characteristic of mRNA of HeLa cells (14). Both fractions contained some large molecular weight RNA which sedimented near the bottom of the gradient. These gradients were characteristic of RNA obtained from cells labeled in the presence of an inhibitor of ribosomal RNA (rRNA) synthesis. They showed that the fractionation on oligo(dT)-cellulose was effective. tRNA, which does not contain poly(A) sequences, was not retained by the column, whereas RNA with the characteristics of mRNA and presumably containing poly(A) sequences was retained and could be eluted with a buffer not containing NaCl. Fractionation
of Duck Reticulocyte
mRNA
We have frationated by the same method polysomes obtained from duck reticulocytes. In this case we followed the Az6,, of the material eluted (Fig. 2). In separate experiments 7-16 mg of polysomes were fractionated; 94-96% of the RNA was eluted in peak 1, and 4-6% eluted in peak 2. The RNA of each peak was analyzed by sucrose gradient centrifugation (Fig. 3). Peak 1 contained rRNA and tRNA; peak 2 contained some rRNA and globin mRNA, sedimenting at 10 s
, \,J 10 FRACTION
20
FIG. 2. Chromatography of duck reticulocyte RNA. The RNA eluted in peaks 1 and 2 was precipitated with ethanol for further analysis. The arrow denotes the start of the low salt elution.
22
PEMBERTON,
LIBERTI
AND BAGLIONI
P
0 ? 2 a P
FIG. 3. Analysis by centrifugation on sucrose gradients of the RNA eluted in peak I (panel A) and 2 (panel B) of Fig. 2. Panel C shows a sample of the RNA of duck reticulocytes before chromatography on oligo(dT)-cellulose; note the presence of the 10 s component and its absence in the pattern shown in panel A. The RNA precipitated from peaks 1 and 2 of Fig. 2 was dissolved in SDS buffer (see Methods); l/20 of the RNA of peak 1 and all the RNA from peak 2 were applied to 36-ml gradients. These were centrifuged 20 hr at 27,000 rpm; 28 s rRNA sedimented to the bottom of the tubes during this time. The RNA of fractions a, b and c of the gradient in panel B was precipitated with ethanol and assayed for translation (Table 1).
(7). In addition, a peak sedimenting at 20 s (20.0 2 0.5 s in four determinations) was observed. In other experiments it was established that peak 2 contains both species of rRNA in approximately the same ratio as peak 1. Globin mRNA should represent 1.7% of the RNA in polysomes made up of an average of five ribosomes (the predominant class of polysomes in our preparations of duck reticulocytes); this value is calculated from the molecular weight of rRNA (15) and duck globin mRNA (7). Since the RNA eluted in peak 2 is three times the theoretical amount of globin mRNA present and is contaminated by rRNA, we have concluded that further fractionations are necessary to isolate pure mRNA. The sucrose gradient fractionation of RNA of peak 2 showed a 20 s component; in four different preparations the 20 s peak was found to be higher than or equal to the 18 s peak and always higher than the 10 s peak. We have investigated the nature of the 20 s RNA by translation in an ascites cell-free system dependent on the addition of exogenous mRNA (11). The results obtained (Table 1) indicate that 10 and 20 s RNA are about equally active in stimulating the cell-free system, if the contamination of 20 s RNA with 18 s rRNA is taken into account. The RNA’s have been assayed at a concentration well within the range of a linear response of the ascites cell-free system (11). In addition, the prod-
23
ISOLATION OF mRNA TABLE 1 TRANSLATION OF DUCK RETWJLOCYTE RNA FRACTIONATED nv OLIGO(DT)-CELLULOSE CHROMATOGRAPHY AND GRADIENT CENTRIFUGATION~ RNA fraction (Fig. 3B)
Cpm
Stimulation (cpm + RNAlcpm - RNA)
a) 20 s b) 18s
2,616 1,426
c) 10 s
4,201
No added RNA 10 s RNA. phenol extracted
1,036
-
3.912
3.8
2.5 1.4 4.1
(LEach incubation mixture contained 5 &i of [ 35S]methionine and 2 ~1 of RNA solution (1 mg/ml) in a final vol of 150 ~1. The components of the ascites cell-free system have been described in detail previously (11). After a 30-min incubation at 3O”C, duplicate $1 aliquots were processed for counting; the cpm reported are the average of these samplings. The 10 s RNA, phenol extracted, has been prepared by the iso-amyl alcohol-chloroform procedure (18) from duck reticulocyte polysomes and fractionated by sucrose gradient sedimentation (1 I).
uct synthesized has been analyzed by electrophoresis on 10% acrylamide gels (Fig. 4). Both 10 and 20 s RNA were translated into a protein that migrated as authentic duck globin. This indicated that these RNA’s contain the information for the synthesis of a protein of identical M,, which is most likely duck globin. The stimulation of the ascites cell-free system by duck globin mRNA is less than that reported previously for rabbit globin mRNA (11). The stimulation observed (Table 1) is in the same range as that obtained with duck globin mRNA fractionated by phenol extraction and sucrose gradient centrifugation (3, 11). This relatively low stimulation is a characteristic of duck globin mRNA, which has a low ability to compete with endogenous mRNA (16) and has a different quantitative requirement for at least one initiation factor (17). Moreover, the use of methionine, which is present in very low amount in globin (18), as labeled amino acid causes a lower stimulation of the cell-free system. The advantage of using this amino acid is the very high specific activity at which it can be obtained. The possibility that the template activity of the mRNA prepared by the method described above is reduced by the presence of residual NaCl is unlikely in view of the fact that the RNA is fractionated on sucrose gradients before the translation assay and that 0.1 M NaCl is routinely added to gradient fractions in order to precipitate RNA with ethanol. The RNA in the 20 s peak is an aggregate, since after treatment with dimethylsulphoxide (19) the predominant component observed sediments identically to 10 s globin mRNA. The M, of the 20 s RNA has
24
PEMBERTON,
LIBERTI
AND BAGLIONI
6
20
40
60
60
20
40
60
80
FRACTIONS
FIG. 4. Analysis by electrophoresis on 10% acrylamide gels of the cell-free product of 10 and 20 s RNA (see Fig. 3). Aliquots, 50 ~1, of the incubation mixtures described in Table 1 were treated for 30 min at 30°C with 10 ~1 of ribonuclease A (1 mglml) and dialyzed afterwards against 0.1% Na dodecyl sulfate, 0.2% mercaptoethanol. The samples were applied to gels and the electrophoresis and counting of gel fractions performed as previously described (13). (A), Protein synthesized by duck reticulocytes in the presence of [W]methionine (see Methods); note that some high molecular weight protein migrating more slowly than the main peak of globin is resolved by the electrophoresis; (B) incubation without added RNA; (C), incubation with added 20 s RNA; (D), incubation with added 10 s RNA.
been estimated from its sedimentation to be 8.5 X 105; this is very close to the M, expected for a complex of mRNA and 18 s rRNA. A similar aggregate has recently been reported by Macnaughton et al. (20). However, the results of disaggregation with dimethylsulfoxide suggest an aggregate of four molecules of duck globin mRNA (M, = 210,000; ref. 7), a possibility also considered by Macnaughton et al. (20). The reasons for the formation of an aggregate of this type are not clear. It has recently been shown that the presence of NaCl in buffers used for the extraction of nuclear RNA leads to the formation of aggregates, which can be reversibly disaggregated (2 1). The presence of 0.5 M NaCl in our starting buffer may thus favor the aggregation of mRNA. However, a ,
ISOLATION
OF mRNA
25
mRNA aggregate sedimenting at 19 s and coding for light chain in the ascites cell-free system has been observed in a preparation of myeloma RNA eluted from oligo(dT)-cellulose columns with buffers containing KC1 (2). Thus, it is possible that, in general, solutions of high salt concentrations, and particularly NaCl, cause RNA aggregation. The presence of the 20 s aggregate makes it somewhat difficult to compare directly the yields of mRNA obtained by phenol extraction and repeated sucrose gradient fractionation (3,ll) with that obtained by oligo(dT)-cellulose chromatography followed by sucrose gradient fractionation. In the first procedure, any 20 s aggregate present is discarded after the first sucrose gradient, and its observation in such large amounts has only been made possible by the preliminary fractionation on oligo(dT)-cellulose. In terms of yields of 10 s globin mRNA we have consistently obtained comparable results with the two procedures; in terms of total template activity recovered we have obtained somewhat better results with the procedure described in this report. It is, however, difficult to quantitate exactly the yield of mRNA by either of these procedures. The losses incurred in the precipitation of RNA in the course of repeated sucrose gradient fractionations are, for instance, not precisely known. Fractionation
of Histone mRNA from HeLa Cells
Histone mRNA does not contain the poly(A) sequence present in other mRNA species (4). It was thus of interest to follow its fractionation by oligo(dT)-cellulose chromatography. Histone mRNA can be labeled with [3H]uridine in the presence of 40 rig/ml of actinomycin (27) and can be identified by electrophoresis on acrylamide gels, where it migrates as a 7-9 s RNA species (22). The identity of this mRNA has been established by translation in a cell-free system (13). Treatment of the cells with inhibitors of DNA synthesis causes the disappearance of histone mRNA (23). RNA extracted from polysomes obtained from HeLa cells incubated with [3H]uridine, as described above, was dissolved in starting buffer and fractionated by chromatography on oligo(dT)-cellulose. The RNA in peaks 1 and 2 was precipitated with ethanol and analyzed by electrophoresis on 3% acrylamide gels (Fig. 5). Histone mRNA was eluted with peak 1, together with rRNA, tRNA, 5 s RNA and some high molecular weight material, which barely migrated into the gel (Fig. 5C). Peak 2 showed several unresolved species of RNA, which gave a broad peak on electrophoresis; histone mRNA was absent from peak 2. As a control, the RNA of HeLa cells, treated for 30 min with 25 pug/ml of cytosine arabinoside to inhibit DNA synthesis (23), was analyzed by electrophoresis on acrylamide gels. The 9 s peak was absent from this RNA;
26
PEMBERTON,
LIBERTI
AND
BAGLIONI
FRACTIONS
5. Analysis by electrophoresis on 3% acrylamide gels of the HeLa cell RNA fractionated by oligo(dT)-cellulose. The conditions for the labeling of the cells, the preparation of polysomes and RNA, and the chromatography on oligo(dT)-cellulose are described in Methods. (A), unfractionated RNA; (B), RNA fraction eluted in peak 2; note the absence of the 9 s component; (C) RNA fraction eluted in peak 1. The position of rRNA markers labeled with [‘Wluridine is indicated in A for reference, Identical ahquots were applied in A and B, whereas an aliquot four times larger was applied in C. The gels were run, sliced and counted as described previously (9, 10). FIG.
this finding supported the identification of the 9 s peak as histone mRNA. This discussion of histone mRNA chromatography is confirmatory to a large extent; however, it is important to point out that we have observed in our analysis very little mRNA (as defined by the labeling in the presence of actinomycin) migrating between 9 and 28 s (Fig. 5C). This contrasts with the recent report by Milcarek et al. (24) that about 30% of HeLa cell mRNA lacks poly(A) when labeled in the presence of different rRNA inhibitors. The reasons for this discrepancy are not clear; the possibility that some of the mRNA lacking poly(A) is an aggregate of histone mRNA of the type reported above, though unlikely in view of its kinetics of synthesis (24), has not yet been completely ruled out. The only definitive resolution of this controversial point will come out of translation experiments, which are currently in progress. DISCUSSION
The method described for the isolation of mRNA is rather simple and avoids any extraction with organic solvents. The possibility of shearing RNA molecules is thus minimized. The method is highly reproducible and the fractionation is carried out at room temperature in the presence of a strong detergent, which inactivates ribonuclease. No special precaution has been taken for cleaning the glassware or for handling the RNA samples. The RNA obtained is contaminated with rRNA (Fig. 3). However, this contamination does not interfere with the translation of mRNA in
ISOLATION
OF
mRN A
27
the ascites cell-free system. It has been previously shown that below a given concentration rRNA actually stimulates the translation of rabbit globin mRNA (11). An aggregate of duck globin mRNA is found in the fraction eluted from oligo(dT)-cellulose at low salt concentration. This 20 s aggregate is translated efficiently by an ascites cell-free system. On the basis of its sedimentation after disaggregation with dimethylsulphoxide, it seems possible that the 20 s RNA is an aggregate of four molecules of mRNA. Isolation of discrete mRNA species thus requires treatment of the RNA eluted from oligo(dT)-cellulose with dimethylsulphoxide. The method described can be used for the separation of histone mRNA from other mRNA’s. Since histone mRNA does not contain poly(A) (4) it is not retained by the column used. Further fractionations are necessary to separate histone mRNA from the other RNA species present in peak 1. The high molecular weight RNA which is eluted with peak 1 (Fig. 5C) is most likely nuclear RNA that contaminates the polyribosome preparation. An analysis of heterogenous nuclear RNA has shown that a large portion of the molecules do not contain poly(A) (25,26).
In conclusion, a simple method for the isolation of an RNA fraction highly enriched in mRNA has been described. This method will find numerous applications in the isolation of RNA fractions that are to be tested in translation assays. It can also be used as the initial step for the preparation of discrete species of mRNA. ACKNOWLEDGMENTS This work was supported by Grant No. AI 08116 from NIH and No. GB 14345 from NSF to C.B. and from the National Research Council (Italy).
REFERENCES 1. Aviv. H., and Leder. P. (1972) Proc. Nat. Acad. Sci. USA 69, 1408-1412. 2. Swan, D., Aviv, H., and Leder, P. (1972) Proc. Nat. Acad. Sci. USA 69, 1967-1971. 3. Pew, R. P., La Terre, J., Kelley, D. E.. and Greenberg, J. R. (1972) Biochim. Biophys. Acta 262, 220-226. 4. Adesnik, M., and Darnell. J. E. (1972) J. Mol. Biol. 67, 397-406. 5. Eagle, H. (1959) Science 130, 432-437. 6. Perry, R. P. (1962) Proc. Nat. Acad. Sci. USA 48, 2179-2186. 7. Pemberton, R. E., Housman, D.. Lodish, H. F., and Baglioni, C. (1972) Nature Newa Biol. 235, 99-102. 8. Oda, K., and Joklik, W. K. (1967) .I. Mol. Biol. 27, 395-419. 9. Loening, U. (1967) Biochem. J. 102, 2.51-257. 10. Weinberg, R. A., and Penman, S. (1968) .I. Mol. Bid!. 38, 289-304. t 1. Jacobs-Lorena, M., and Baglioni, C. (1972) Biochemistry 11, 4970-4974. 12. Pemberton, R. E., and Bagloni, C. (1972)J. Mol. Bio/. 65, 531-535. 13. Jacobs-Lorena, M., Baglioni, C., and Borun. T. W. (1972) Proc. Nat. Acad. Sri. USA 69. 2095-2099.
28
PEMBERTON,
LIBERTI
AND BAGLIONI
14. Singer, R. H., and Penman, S. (1972) Nature (London) 240, 100-102. 15. Darnell, J. E. (1968) Bacterial. Rev. 32, 262-290. 16. Stewart, A. G., Gander, E. S., Morel, C., Luppis, B., and Scherrer, K. (1973) Eur. J. Biochem. 34, 205-2 12. 17. Schreier, M. H., Staehelin, T., Stewart, A. G., Gander, E. S., and Scherrer, K. (1973) Eur. J. Biochem. 34, 2 13-218. 18. In Atlas of Protein Sequence and Structure. (Dayhoff, M. O., ed.), National Biomedical Research Foundation, Washington, Vol. 5, 1972. 19. Lindberg, U., and Darnell, J. E. (1970) Proc. Nar. Acad. Sci. USA 65, 1089-1096. 20. Macnaughton, M., Freeman, K. B., and Bishop, J. 0. (1974) Cc/l 1, 117-I 26. 21. Bramwell, M. E. (1972) Biochim. Biophys. Acta 281, 329-337. 22. Schochetman, G., and Perry, R. P. (1972) J. Mol. Biol. 63, 591-596. 23. Borun, T. W., ScharlT, M. D., and Robbins, E. (1967) Proc. Nat. Acad. Sci. USA 68, 1321-1325. 24. Milcarek, C., Proce, R., and Penman, S. (1974) Cell 3, l-10. 25. Darnell, J. E., Wall, R., and Tushinski, R. J. (1971) Proc. Nat. Acad. Sci. USA 68, 1321-1325. 26. Greenberg, J. R., and Perry, R. P. (1972) 1. Mol. Biol. 72, 91-98.
BIOCHEMISTRY
Isolation
66, 18-28 (1975)
of Messenger
by Chromatography R.E.
RNA
from
Polysomes
on Oligo(dT)-Cellulose
PEMBERTON, P. LIBERTI, AND C. BACLIONI
Department Cambridge,
of Biology, Massachusetts Institute of Technology, Massachusetts, and Laboratory of Cell Biology, via Romagnosi 18/A, Rome, Italy
Received April 15, 1974; accepted December 24, 1974 A method for the isolation of messenger RNA (mRNA) from polysomes is described. Pofysomes are dissolved in a solution containing0.5 M NaCl and Na dodecyl sulphate and applied to an oligo(dT)-cellulose column. RNA speciescontaining poly(A) sequences are retained by the column, whereas ribosomal proteins and other RNA species are washed off. The column is then eluted with a buffer not containing NaCI. mRNA from HeLa cells and from duck reticulocytes has been fractionated in this way. When fractionated on sucrose gradients, 10 s globin mRNA is obtained in addition to a 20 s component, which can be translated in a cell free system into duck globin. This 20 s RNA is an aggregate of mRNA, which can be disaggregated. Experiments with HeLa cells have shown that the only mRNA species which is not retained by oligo(dT)-cellulose is histone mRNA; this mRNA does not contain a poly(A) sequence.
Rabbit globin mRNA has been isolated by Aviv and Leder (1) from rabbit reticulocyte RNA by chromatography on oligo(dT)-cellulose. Subsequently, mRNA for the light chain of a mouse immunoglobulin has been isolated by the same method from the RNA of a myeloma and its identity established by translation in an ascites cell-free system (2). This chromatographic method can thus be generally used for the isolation of mRNA’s containing poly(A) sequences. So far, phenol extraction has been used for the preliminary extraction of RNA from polysomes (1,2). Phenol extraction presents some disadvantages, particularly the risk of losing part or all of the poly(A) sequence (3). This can be avoided by using extraction procedures that have been shown to preserve the integrity of poly(A) (3). It would be preferable, however, to avoid any extraction and to isolate mRNA by mild procedures. We have thus isolated mRNA by direct chromatography of resuspended polysomes on oligo(dT)-cellulose. The polysomes are dissociated by the use of the strong detergent Na dodecyl sulphate which is present throughout the chromatography. The RNA isolated has been analyzed by sucrose gradients and aggregation of duck globin mRNA into a discrete 20 s component has been noticed. The formation of this 18 Copyright Al, &“htr
@ 1975 by Academic d*a..-^rl ..^. :.- ‘-
Press, Inc. -
*
ISOLATION
aggregate and the behavior of histone mRNA, poly(A) sequences (4), have also been studied. MATERIALS
19
OF mRNA
AND
which does not contain
METHODS
Column chromatography on oligo(dT)-cellulose. A 0.5 X 4-cm cdumn of oligo(dT)-cellulose, supplied by Collaborative Research Inc., was equilibrated with 0.5 M NaCl, 0.5% Na dodecyl sulphate and 0.01 M Tris buffer, pH 7.5 (starting buffer). The samples applied to the column were dissolved in water and then diluted with an equal volume of twice-concentrated starting buffer. The column was washed with approximately 30 ml of starting buffer and then eluted with 0.5% Na dodecyl sulphate, 0.01 M Tris buffer, pH 7.5. All the operations, after the addition of starting buffer to the samples, were carried out at room temperature. RNA was precipitated with two volumes of ethanol at 20°C after adjusting the NaCl concentration to 0.1 M. Polysomes. HeLa cells were grown in minimal Eagle’s medium (5) in spinner bottles. The cells were collected by centrifugation and resuspended at 2 X 10 cell/ml. The cells were incubated for 30 min with 40 rig/ml of actinomycin D to inhibit the synthesis of ribosomal RNA (6) and then for 2 hr with 40 &i/ml of [5-3H]uridine (56.7 Cilmole; New England Nuclear). The cells were collected by centrifugation, washed with ice-cold Earle’s saline and resuspended in RSB buffer (10 mM NaCl, 10 mM Tris, pH 7.4, and 1.5 mM MgCl,; I ml of buffer/lo7 cells). After 10 min the cells were homogenized and the postmitochondrial supernatant obtained by centrifugation for IO min at 20,000 g. This was applied over 30% sucrose in RSB and the polysomes pelletted by a 3-hr centrifugation at 150,OOOg. Duck reticulocytes were obtained as previously described (7) and reticulocyte polysomes prepared as described above. Preparation and analysis of RNA. Polysome pellets were dissolved in SDS buffer (0.1 M NaCl, 10 mM Tris pH 7.4, 10 mM ethylene diamino tetraacetic acid and 0.5% Na dodecyl sulphate) to extract RNA as previously described (8). The polysome pellets were dissolved in starting buffer (see above) for fractionation of the RNA by oligo(dT)-cellulose chromatography. The RNA obtained by either method was precipitated with ethanol and further characterized by sucrose gradient centrifugation or acrylamide-gel electrophoresis. Centrifugations were run as described in the legends to figures, using 5-20% sucrose gradients in SDS buffer. Fractions were counted after precipitation with 5% trichloroacetic acid. For the electrophoretic analysis 3% acrylamide gels were run for 150 min, sliced and counted as previously described (9,lO). RNA fractions were assayed in the ascites cell free system described in detail previously (11).
20
PEMBERTON,
LIBERTI
AND BAGLIONI
Preparation and analysis of proteins. HeLa cell histones uniformly labeled with [14C]lysine were prepared as described previously (13). Duck hemoglobin uniformly labeled with [35S]methionine was prepared by incubating 1 ml of duck reticulocytes for 18 hr with 100 #Zi of [35S]methionine (90 Cilmole; New England Nuclear) as previously described (12); the cells were lysed and the postmitochondrial supematant fraction prepared as described above. This protein and the product of the cell-free system were analyzed by electrophoresis on 10% acrylamide gels. These were prepared, run, fractionated and counted as previously described ( 13).
RESULTS Fractionation
of HeLa Cells mRNA
In order to fractionate RNA on oligo(dT)-cellulose columns without prior phenol extraction, we have used buffers containing the detergent Na dodecyl sulphate to dissolve polysomes and have included the same detergent in the buffers used to equilibrate and elute the columns. It was thus necessary to substitute the KC1 used previously (1,2) with NaCl to avoid precipitation of the detergent. In a preliminary experiment we established that even very basic proteins, like histones, are not absorbed by oligo(dT)-cellulose. In this experiment we mixed 0.03 mg of HeLa cells RNA labeled with C3H]uridine in the presence of actinomycin D and extracted from polysomes (see Methods) with 0.01 mg of HeLa cell histones labeled with [r4C]lysine; 1 mg of yeast tRNA was added as carrier to allow us to follow the AzeO pattern. In the chromatography on oligo(dT)-cellulose (not shown), 98% of the histones applied were recov-
FRACTIONS
FIG. 1. Chromatography of HeLa cell RNA on oligo(dT)-cellulose. (A), Elution pattern of the RNA labeled with [3H]uridine in presence of 40 rig/ml of actinomycin D (see Methods). The arrow denotes the start of the low salt elution. Two-milliliter fractions were collected; (B), analysis by centrifugation on sucrose gradients of the RNA eluted in peaks 1 and 2 of A. The patterns obtained in two separate gradients are superimposed and the position of marker RNA species run on parallel gradients is shown for reference. The gradients were centrifuged for 14 hr at 25,000 rpm.
ISOLATION
OF mRNA
21
ered in peak 1 (see Fig. I), and nearly 100% of the HeLa RNA was recovered, 44% in peak 1 and 66% in peak 2 (the fraction eluted with buffer not containing NaCl; see Methods and Fig. 1A). Polysomes prepared from HeLa ceils labeled with [3H]uridine were dissolved in starting buffer and fractionated by chromatography on oligo(dT)-cellulose (Fig. 1A). Again, the RNA was found partitioned, 38% in peak 1 and 62% in peak 2. The fractions corresponding to each peak were pooled and the RNA precipitated and analyzed by sucrose gradient centrifugation (Fig. 1B). The RNA of peak 1 consisted mainly of tRNA (4 s RNA), whereas the RNA of peak 2 sedimented in a rather broad peak, characteristic of mRNA of HeLa cells (14). Both fractions contained some large molecular weight RNA which sedimented near the bottom of the gradient. These gradients were characteristic of RNA obtained from cells labeled in the presence of an inhibitor of ribosomal RNA (rRNA) synthesis. They showed that the fractionation on oligo(dT)-cellulose was effective. tRNA, which does not contain poly(A) sequences, was not retained by the column, whereas RNA with the characteristics of mRNA and presumably containing poly(A) sequences was retained and could be eluted with a buffer not containing NaCl. Fractionation
of Duck Reticulocyte
mRNA
We have frationated by the same method polysomes obtained from duck reticulocytes. In this case we followed the Az6,, of the material eluted (Fig. 2). In separate experiments 7-16 mg of polysomes were fractionated; 94-96% of the RNA was eluted in peak 1, and 4-6% eluted in peak 2. The RNA of each peak was analyzed by sucrose gradient centrifugation (Fig. 3). Peak 1 contained rRNA and tRNA; peak 2 contained some rRNA and globin mRNA, sedimenting at 10 s
, \,J 10 FRACTION
20
FIG. 2. Chromatography of duck reticulocyte RNA. The RNA eluted in peaks 1 and 2 was precipitated with ethanol for further analysis. The arrow denotes the start of the low salt elution.
22
PEMBERTON,
LIBERTI
AND BAGLIONI
P
0 ? 2 a P
FIG. 3. Analysis by centrifugation on sucrose gradients of the RNA eluted in peak I (panel A) and 2 (panel B) of Fig. 2. Panel C shows a sample of the RNA of duck reticulocytes before chromatography on oligo(dT)-cellulose; note the presence of the 10 s component and its absence in the pattern shown in panel A. The RNA precipitated from peaks 1 and 2 of Fig. 2 was dissolved in SDS buffer (see Methods); l/20 of the RNA of peak 1 and all the RNA from peak 2 were applied to 36-ml gradients. These were centrifuged 20 hr at 27,000 rpm; 28 s rRNA sedimented to the bottom of the tubes during this time. The RNA of fractions a, b and c of the gradient in panel B was precipitated with ethanol and assayed for translation (Table 1).
(7). In addition, a peak sedimenting at 20 s (20.0 2 0.5 s in four determinations) was observed. In other experiments it was established that peak 2 contains both species of rRNA in approximately the same ratio as peak 1. Globin mRNA should represent 1.7% of the RNA in polysomes made up of an average of five ribosomes (the predominant class of polysomes in our preparations of duck reticulocytes); this value is calculated from the molecular weight of rRNA (15) and duck globin mRNA (7). Since the RNA eluted in peak 2 is three times the theoretical amount of globin mRNA present and is contaminated by rRNA, we have concluded that further fractionations are necessary to isolate pure mRNA. The sucrose gradient fractionation of RNA of peak 2 showed a 20 s component; in four different preparations the 20 s peak was found to be higher than or equal to the 18 s peak and always higher than the 10 s peak. We have investigated the nature of the 20 s RNA by translation in an ascites cell-free system dependent on the addition of exogenous mRNA (11). The results obtained (Table 1) indicate that 10 and 20 s RNA are about equally active in stimulating the cell-free system, if the contamination of 20 s RNA with 18 s rRNA is taken into account. The RNA’s have been assayed at a concentration well within the range of a linear response of the ascites cell-free system (11). In addition, the prod-
23
ISOLATION OF mRNA TABLE 1 TRANSLATION OF DUCK RETWJLOCYTE RNA FRACTIONATED nv OLIGO(DT)-CELLULOSE CHROMATOGRAPHY AND GRADIENT CENTRIFUGATION~ RNA fraction (Fig. 3B)
Cpm
Stimulation (cpm + RNAlcpm - RNA)
a) 20 s b) 18s
2,616 1,426
c) 10 s
4,201
No added RNA 10 s RNA. phenol extracted
1,036
-
3.912
3.8
2.5 1.4 4.1
(LEach incubation mixture contained 5 &i of [ 35S]methionine and 2 ~1 of RNA solution (1 mg/ml) in a final vol of 150 ~1. The components of the ascites cell-free system have been described in detail previously (11). After a 30-min incubation at 3O”C, duplicate $1 aliquots were processed for counting; the cpm reported are the average of these samplings. The 10 s RNA, phenol extracted, has been prepared by the iso-amyl alcohol-chloroform procedure (18) from duck reticulocyte polysomes and fractionated by sucrose gradient sedimentation (1 I).
uct synthesized has been analyzed by electrophoresis on 10% acrylamide gels (Fig. 4). Both 10 and 20 s RNA were translated into a protein that migrated as authentic duck globin. This indicated that these RNA’s contain the information for the synthesis of a protein of identical M,, which is most likely duck globin. The stimulation of the ascites cell-free system by duck globin mRNA is less than that reported previously for rabbit globin mRNA (11). The stimulation observed (Table 1) is in the same range as that obtained with duck globin mRNA fractionated by phenol extraction and sucrose gradient centrifugation (3, 11). This relatively low stimulation is a characteristic of duck globin mRNA, which has a low ability to compete with endogenous mRNA (16) and has a different quantitative requirement for at least one initiation factor (17). Moreover, the use of methionine, which is present in very low amount in globin (18), as labeled amino acid causes a lower stimulation of the cell-free system. The advantage of using this amino acid is the very high specific activity at which it can be obtained. The possibility that the template activity of the mRNA prepared by the method described above is reduced by the presence of residual NaCl is unlikely in view of the fact that the RNA is fractionated on sucrose gradients before the translation assay and that 0.1 M NaCl is routinely added to gradient fractions in order to precipitate RNA with ethanol. The RNA in the 20 s peak is an aggregate, since after treatment with dimethylsulphoxide (19) the predominant component observed sediments identically to 10 s globin mRNA. The M, of the 20 s RNA has
24
PEMBERTON,
LIBERTI
AND BAGLIONI
6
20
40
60
60
20
40
60
80
FRACTIONS
FIG. 4. Analysis by electrophoresis on 10% acrylamide gels of the cell-free product of 10 and 20 s RNA (see Fig. 3). Aliquots, 50 ~1, of the incubation mixtures described in Table 1 were treated for 30 min at 30°C with 10 ~1 of ribonuclease A (1 mglml) and dialyzed afterwards against 0.1% Na dodecyl sulfate, 0.2% mercaptoethanol. The samples were applied to gels and the electrophoresis and counting of gel fractions performed as previously described (13). (A), Protein synthesized by duck reticulocytes in the presence of [W]methionine (see Methods); note that some high molecular weight protein migrating more slowly than the main peak of globin is resolved by the electrophoresis; (B) incubation without added RNA; (C), incubation with added 20 s RNA; (D), incubation with added 10 s RNA.
been estimated from its sedimentation to be 8.5 X 105; this is very close to the M, expected for a complex of mRNA and 18 s rRNA. A similar aggregate has recently been reported by Macnaughton et al. (20). However, the results of disaggregation with dimethylsulfoxide suggest an aggregate of four molecules of duck globin mRNA (M, = 210,000; ref. 7), a possibility also considered by Macnaughton et al. (20). The reasons for the formation of an aggregate of this type are not clear. It has recently been shown that the presence of NaCl in buffers used for the extraction of nuclear RNA leads to the formation of aggregates, which can be reversibly disaggregated (2 1). The presence of 0.5 M NaCl in our starting buffer may thus favor the aggregation of mRNA. However, a ,
ISOLATION
OF mRNA
25
mRNA aggregate sedimenting at 19 s and coding for light chain in the ascites cell-free system has been observed in a preparation of myeloma RNA eluted from oligo(dT)-cellulose columns with buffers containing KC1 (2). Thus, it is possible that, in general, solutions of high salt concentrations, and particularly NaCl, cause RNA aggregation. The presence of the 20 s aggregate makes it somewhat difficult to compare directly the yields of mRNA obtained by phenol extraction and repeated sucrose gradient fractionation (3,ll) with that obtained by oligo(dT)-cellulose chromatography followed by sucrose gradient fractionation. In the first procedure, any 20 s aggregate present is discarded after the first sucrose gradient, and its observation in such large amounts has only been made possible by the preliminary fractionation on oligo(dT)-cellulose. In terms of yields of 10 s globin mRNA we have consistently obtained comparable results with the two procedures; in terms of total template activity recovered we have obtained somewhat better results with the procedure described in this report. It is, however, difficult to quantitate exactly the yield of mRNA by either of these procedures. The losses incurred in the precipitation of RNA in the course of repeated sucrose gradient fractionations are, for instance, not precisely known. Fractionation
of Histone mRNA from HeLa Cells
Histone mRNA does not contain the poly(A) sequence present in other mRNA species (4). It was thus of interest to follow its fractionation by oligo(dT)-cellulose chromatography. Histone mRNA can be labeled with [3H]uridine in the presence of 40 rig/ml of actinomycin (27) and can be identified by electrophoresis on acrylamide gels, where it migrates as a 7-9 s RNA species (22). The identity of this mRNA has been established by translation in a cell-free system (13). Treatment of the cells with inhibitors of DNA synthesis causes the disappearance of histone mRNA (23). RNA extracted from polysomes obtained from HeLa cells incubated with [3H]uridine, as described above, was dissolved in starting buffer and fractionated by chromatography on oligo(dT)-cellulose. The RNA in peaks 1 and 2 was precipitated with ethanol and analyzed by electrophoresis on 3% acrylamide gels (Fig. 5). Histone mRNA was eluted with peak 1, together with rRNA, tRNA, 5 s RNA and some high molecular weight material, which barely migrated into the gel (Fig. 5C). Peak 2 showed several unresolved species of RNA, which gave a broad peak on electrophoresis; histone mRNA was absent from peak 2. As a control, the RNA of HeLa cells, treated for 30 min with 25 pug/ml of cytosine arabinoside to inhibit DNA synthesis (23), was analyzed by electrophoresis on acrylamide gels. The 9 s peak was absent from this RNA;
26
PEMBERTON,
LIBERTI
AND
BAGLIONI
FRACTIONS
5. Analysis by electrophoresis on 3% acrylamide gels of the HeLa cell RNA fractionated by oligo(dT)-cellulose. The conditions for the labeling of the cells, the preparation of polysomes and RNA, and the chromatography on oligo(dT)-cellulose are described in Methods. (A), unfractionated RNA; (B), RNA fraction eluted in peak 2; note the absence of the 9 s component; (C) RNA fraction eluted in peak 1. The position of rRNA markers labeled with [‘Wluridine is indicated in A for reference, Identical ahquots were applied in A and B, whereas an aliquot four times larger was applied in C. The gels were run, sliced and counted as described previously (9, 10). FIG.
this finding supported the identification of the 9 s peak as histone mRNA. This discussion of histone mRNA chromatography is confirmatory to a large extent; however, it is important to point out that we have observed in our analysis very little mRNA (as defined by the labeling in the presence of actinomycin) migrating between 9 and 28 s (Fig. 5C). This contrasts with the recent report by Milcarek et al. (24) that about 30% of HeLa cell mRNA lacks poly(A) when labeled in the presence of different rRNA inhibitors. The reasons for this discrepancy are not clear; the possibility that some of the mRNA lacking poly(A) is an aggregate of histone mRNA of the type reported above, though unlikely in view of its kinetics of synthesis (24), has not yet been completely ruled out. The only definitive resolution of this controversial point will come out of translation experiments, which are currently in progress. DISCUSSION
The method described for the isolation of mRNA is rather simple and avoids any extraction with organic solvents. The possibility of shearing RNA molecules is thus minimized. The method is highly reproducible and the fractionation is carried out at room temperature in the presence of a strong detergent, which inactivates ribonuclease. No special precaution has been taken for cleaning the glassware or for handling the RNA samples. The RNA obtained is contaminated with rRNA (Fig. 3). However, this contamination does not interfere with the translation of mRNA in
ISOLATION
OF
mRN A
27
the ascites cell-free system. It has been previously shown that below a given concentration rRNA actually stimulates the translation of rabbit globin mRNA (11). An aggregate of duck globin mRNA is found in the fraction eluted from oligo(dT)-cellulose at low salt concentration. This 20 s aggregate is translated efficiently by an ascites cell-free system. On the basis of its sedimentation after disaggregation with dimethylsulphoxide, it seems possible that the 20 s RNA is an aggregate of four molecules of mRNA. Isolation of discrete mRNA species thus requires treatment of the RNA eluted from oligo(dT)-cellulose with dimethylsulphoxide. The method described can be used for the separation of histone mRNA from other mRNA’s. Since histone mRNA does not contain poly(A) (4) it is not retained by the column used. Further fractionations are necessary to separate histone mRNA from the other RNA species present in peak 1. The high molecular weight RNA which is eluted with peak 1 (Fig. 5C) is most likely nuclear RNA that contaminates the polyribosome preparation. An analysis of heterogenous nuclear RNA has shown that a large portion of the molecules do not contain poly(A) (25,26).
In conclusion, a simple method for the isolation of an RNA fraction highly enriched in mRNA has been described. This method will find numerous applications in the isolation of RNA fractions that are to be tested in translation assays. It can also be used as the initial step for the preparation of discrete species of mRNA. ACKNOWLEDGMENTS This work was supported by Grant No. AI 08116 from NIH and No. GB 14345 from NSF to C.B. and from the National Research Council (Italy).
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AND BAGLIONI
14. Singer, R. H., and Penman, S. (1972) Nature (London) 240, 100-102. 15. Darnell, J. E. (1968) Bacterial. Rev. 32, 262-290. 16. Stewart, A. G., Gander, E. S., Morel, C., Luppis, B., and Scherrer, K. (1973) Eur. J. Biochem. 34, 205-2 12. 17. Schreier, M. H., Staehelin, T., Stewart, A. G., Gander, E. S., and Scherrer, K. (1973) Eur. J. Biochem. 34, 2 13-218. 18. In Atlas of Protein Sequence and Structure. (Dayhoff, M. O., ed.), National Biomedical Research Foundation, Washington, Vol. 5, 1972. 19. Lindberg, U., and Darnell, J. E. (1970) Proc. Nar. Acad. Sci. USA 65, 1089-1096. 20. Macnaughton, M., Freeman, K. B., and Bishop, J. 0. (1974) Cc/l 1, 117-I 26. 21. Bramwell, M. E. (1972) Biochim. Biophys. Acta 281, 329-337. 22. Schochetman, G., and Perry, R. P. (1972) J. Mol. Biol. 63, 591-596. 23. Borun, T. W., ScharlT, M. D., and Robbins, E. (1967) Proc. Nat. Acad. Sci. USA 68, 1321-1325. 24. Milcarek, C., Proce, R., and Penman, S. (1974) Cell 3, l-10. 25. Darnell, J. E., Wall, R., and Tushinski, R. J. (1971) Proc. Nat. Acad. Sci. USA 68, 1321-1325. 26. Greenberg, J. R., and Perry, R. P. (1972) 1. Mol. Biol. 72, 91-98.
