[56] The isolation of messenger RNA from mammalian cells

[56]

THE ISOLATION OF MESSENGER R N A

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The Isolation of Messenger Mammalian Cells 1

RNA

605

from

By GEORGE BRAWERMAN Criteria for the Identification o] m R N A

Recent advances in our knowledge of mammalian messenger RNA have permitted a rational approach to its identification and isolation. Until recently, the only criteria generally available for the detection of mRNA in animal cells were: (a) presence in polysomes; (b) rapid labeling; (c) heterogeneity in size distribution. DNA-like base composition is used sometimes as an additional criterion. In order to distinguish between polysomal R N A and other R N A species that might cosediment with the polysomes, some investigators have dissociated the polysomes with E D T A and utilized only those RNA components released as more slowly sedimenting material by this treatment. 2 The preferential inhibition of ribosomal RNA synthesis by actinomycin D has provided a convenient means for avoiding the presence of ribosomal label in the polysomes. In many mammalian cell cultures, actinomycin levels of 0.04 ~g/ml are sufficient to block rRNA synthesis, but have little effect on the labeling of the presumed mRNA. -~,3 The above criteria provide a clear distinction between ribosomal and nonribosomal RNA, but fail to discriminate effectively between mRNA and other R N A components that might contaminate the polysome preparations. Such contaminants could cause difficulties in cases where highly labeled nuclear R N A has leaked from the nuclei during cell disruption. '-',4 The development of mammalian cell-free systems that will synthesize defined proteins in response to exogenous R N A has provided a precise means for identifying unique m R N A species. The messenger for hemoglobin, for instance, has been well characterized by its sedimentation coefficient of 9 - 1 0 S and its capacity to induce globin synthesis in heterologous cell-free systems. '~." Other mRNA species have been assayed in a similar fashion. 'Supported by a research grant from the U.S. Public Health Service (GM 17973). "S. Penman, C. Vesco, and M. Penman, J. Mol. Biol. 34, 49 (1968). 3R. Perry, Exp. Cell Res. 29, 400 (1963). 4R. P. Perry and D. E. Kelley, 1. Mol. Biol. 35, 37 (1968). 5 R. E. Lockard and J. B. Lingrel, Biochem. Biophys. Res. Commun. 37, 204 (1969). "D. Housman, R. Pemberton, and R. Taber, Proc. Nat. Acad. Sci. U.S. 68, 2716 (1972).

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MESSENGERR N A AND PROTEIN SYNTHESIZING SYSTEMS

Presence o / P o l y ( A )

[56]

in m R N A

It has been found recently that most of the heterodisperse nonribosomal polysomal R N A of mammalian cells contains a polyadenylate sequence at the 3' end of the molecules. 7-1° The occurrence of p o l y ( A ) in mRNA may be a characteristic of all eukaryotic cells. The poly(A) segment has been detected in most of the mRNA species active as templates for synthesis of specific proteins. The m R N A for histones, however, has been shown to lack the p o l y ( A ) sequence. 11 The presence of p o l y ( A ) in most mRNA, and its absence from ribosomal and transfer RNA, provides a precise criterion for the identification of mRNA in polysomes. P o l y ( A ) possesses unique properties, such as capacity to form complementary base-paired structures with p o l y ( U ) and p o l y ( d T ) and to bind to cellulose nitrate membrane filters (Millipore filters) at high ionic strength. These properties provide simple means for the separation of m R N A from other R N A species. Radioactive mRNA can be assayed most conveniently by its capacity to bind to Millipore filters, T and the labeling need not be done in the presence of actinomycin, since labeled RNA components other than mRNA are not retained on the filters. P o l y ( A ) also exhibits a unique behavior during deproteinization with phenol. It appears to bind to denatured proteins in the presence of monovalent cations, and, as a result, is carried to the nonaqueous phase. 12 Because of this property of the p o l y ( A ) segment, mRNA may not be readily extracted from biological preparations, even under conditions that favor the recovery of the bulk of the cellular RNA. It has been possible, however, to define some of the parameters that control the apparent p r o t e i n - p o l y ( A ) interaction in the phenol mixture, and to establish optimal conditions for the extraction of mRNA. 1'-' Extraction of m R N A Resistance o] m R N A

to Phenol Extraction

It had been known for some time that mammalian cells contain an R N A fraction refractory to phenol extraction. Aqueous phenol treatment 7S. Y. Lee, J. Mendecki, and G. Brawerman, Proc. Nat. Acad. Sci. U.S. 68, 1331 (1971). s M. Edmonds, M. H. Vaughan, and H. Nakazato, Proc. Nat. Acad. Sci. U.S. 68, 1336 (1971). 9j. E. Darnell, R. Wall, and R. J. Tushinski, Proc. Nat. ,4cad. Sci. U.S. 68, 1321 (1971). loj. Mendecki, S. Y. Lee, and G. Brawerman, Biochemistry 11, 792 (1972). riM. Adesnik and J. E. Darnell, J. Mol. Biol. 67, 397 (1972). 12G. Brawerman, J. Mendecki, and S. Y. Lee, Biochemistry 11, 637 (1972).

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THE ISOLATION OF MESSENGER R N A

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TABLE I PHENOL FRACTIONATION OF SARCOMA 1~0 POLYSOMES a

RNA fraction

Total R N A in fraction (ug)

Radioactivity (cpm)

Millipore-bound radioactivity (~. of total in fraction)

pH 7.6 pH 9

1152 38

54,500 79,250

2 100

Polysomes from cells labeled with uridine in presence of 0.04 ug/ml actinomyein D were subjected to phenol extraction in presence of 0.1 M Tris.HC1 (pH 7.6). Nonaqueous residue reextracted first with 0.1 M Tris (pH 7.6) then twice with 0.1 M Tris (pH 9.0). R N A purified from pooled neutral and alkaline extracts. D a t a derived from (L Brawerman, J. Mendecki, and S. Y. Lee, Biochemistry t l , 637 (1972).

at neutral pH in the cold of homogenized liver and of disrupted ascites cells was shown to result in the release of the ribosomal RNA into the aqueous phase, but to leave a minor RNA fraction in the nonaqueous phase. ''~ The latter could be recovered by reextraction at high temperature. It was shown to be DNA-like in base composition. A similar RNA fraction could be recovered from the nonaqueous phase by reextraction with slightly alkaline Tris buffer?' The latter fraction shall be referred to as pH 9 RNA (RNA not extracted into the aqueous phase in the presence of Tris.HCl pH 7.6, but recoverable with Tris pH 9). This RNA fraction was found to be present in purified polysomes from mouse sarcoma 180 ascites cells5 It was further observed that the poly(A) sequence was present in the polysomal pH 9 RNA, and absent from the RNA extractable at neutral pH. Table I shows the distribution in the two phenol fractions of the bulk polysomal RNA and of the material labeled in the presence of a low level of actinomycin. Only the labeled RNA extracted at pH 9 contains poly(A), as indicated by its capacity to bind to Millipore filters.

Conditions for the Phenol Extraction o] mRNA The behavior of the pH 9 RNA during phenol extraction can be accounted for by the properties of the poly(A) segment. It was observed that synthetic poly(A), when mixed with polysomes and subjected to the phenol treatment under the conditions used during RNA extraction

'~ G. P. Georgiev and V. L. Mantieva, Biochim. Biophys. Acta 61, 153 (1962). 1~G. Brawerman, L. Gold, and J. Eisenstadt, Proc. Nat. Acad. Sci. U.S. 50, 630 (1963).

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MESSENGER R N A

AND PROTEIN

SYNTHESIZING SYSTEMS

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TABLE II EFFECT OF POLYSOMES AND OF METHYLATED ALBUMIN ON THE BEHAVIOR OF POLY(A) DURINO PHENOL EXTRACTIONa

Labeled RNA remaining in aqueous phase (% of total) Labeled RNA preparation

No addition

Polysomes added

Methylated albumin added

Poly(A) Polysomal RNA, pH 9 Polysomal RNA, pH 7.6

96 98 --

3 19 68

6 35 80

a Labeled RNA preparations mixed with unlabeled polysomes or methylated albumin and subjected to phenol treatment in 0.1 M Tris.HCl (pH 7.6). Data derived from G. Brawerman, J. Mendecki, and S. Y. Lee, Biochemistry 11,637 (1972).

at p H 7.6, is removed from the aqueous phase (Table I I ) . This appears to be caused by interaction between the p o l y ( A ) and denatured ribosomal proteins. The same effect is observed when methylated albumin is substituted for the ribosomal proteins. Deproteinized p H 9 polysomal R N A shows a similar behavior, in contrast to the p H 7.6 R N A fraction. TM The above interaction between p o l y ( A ) and protein is promoted by monovalent cations. Since Tris is nearly completely protonated at neutral pH, a 0.1 M solution of this buffer contributes a concentration of monovalent cations close to 0.1 M. K ~ and Mg -~÷, included in the medium used to suspend polysomes, are also present. Reextraction of the p H 9 R N A from the nonaqueous residue after p H 7.6 extraction as in Table I, takes place in a considerably reduced ionic environment, since Tris at p H 9.0 occurs primarily in the nonionized form. Inclusion of N a + or K ÷ at this stage interferes with the p H 9 extraction. It appears that the p H 9 R N A can be extracted at neutral p H in the absence of cations, but Tris buffer of p H 9.0 seems more effective and has been used consistently in this laboratory. Procedure for R N A Extraction 12 Reagents Tris buffer, p H 9 . 0 : 1 M solution methane (Tris) neutralized with room temperature and diluted as Aqueous phenol: redistilled phenol tration of 80% ( v / v ) ; stored in

of tris(hydroxymethyl)aminoHC1 to p H 9.0 (measured at required) diluted with H~O to a concenthe cold in a brown bottle

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THE ISOLATION OF MESSENGER R N A

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SDS: 5% solution of sodium dodecyl sulfate; kept at room temperature Procedure. All operations are carried out at 0-5 °. The polysome suspension is diluted with water to a concentration no greater than 100 A..,~o units per milliliter. One-tenth volumes of 5% SDS and of 1 M Tris, pH 9.0, are added, followed by 1 volume of aqueous phenol. The mixture is stirred vigorously for about 5 minutes, then centrifuged at 12,000 g for 10 minutes to separate the phases, and the aqueous phase is removed. The nonaqueous residue (phenol phase plus gel interphase) is reextracted with an equal volume of 0.1 M Tris, pH 9.0, and 0.5% SDS by vigorous stirring as before, and the aqueous phase is removed after centrifugation. The pooled aqueous phases contain most of the polysomal rRNA and mRNA. They are extracted three times with an equal volume of fresh aqueous phenol by brief vigorous stirring followed by centrifugation and removal from the phenol phase. These last extractions with phenol serve to remove residual protein material, as well as most of the SDS. The RNA solution can be used without precipitation, but must be then extracted at least four times with ether to remove dissolved phenol. The ether is subsequently removed by blowing air over the solUtion. The RNA can also be precipitated from the aqueous phase after the last phenol extraction by addition of 0.1 volume of 1 M NaC1 and 2.5 volumes of ethanol. After storage overnight at 4 ° , the precipitate is collected by centrifugation and washed twice with cold 66% ethanol in 0.1 M NaC1, to remove residual phenol. The precipitate can be dissolved in water and stored at - 20 °. Extraction of Nuclear R N A

P o l y ( A ) segments are also present in the nucleoplasmic RNA. These poly(A)-containing nuclear RNA molecules are presumed to represent the precursors to cytoplasmic mRNA. 1°,1~ The pH 9 phenol procedure is effective for their extraction from the nuclei, but DNA is also extracted. The high viscosity of the extracts makes their handling very difficult. Moreover, the R N A cannot be separated easily from the contaminating DNA. Incubation with DNase is necessary to hydrolyze the DNA, and contamination of the enzyme preparation by traces of RNase could easily lead to fragmentation of the R N A molecules during this treatment. The hot SDS-phenol procedure, TM which does not lead to extraction of DNA, appears to be as effective as the pH 9 phenol procedure for the recovery of the poly(A)-containing R N A molecules from nuclei. asj. E. Darnell, L. Philipson, R. Wall, and M. Adesnik, Science 174, 507 (1971). I~M. Girard, this series, Vol. 12A, p. 581.

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MESSENGERRNA AND PROTEIN SYNTHESIZING SYSTEMS

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Other Extraction Procedures for Polysomal mRNA It has been reported that addition of CHCI~ to the phenol leads to the release of poly(A)-containing RNA into the aqueous phase, even when conditions tend to favor the protein-poly(A) interaction. 17 The procedure was found to be effective in this laboratory, with yields of mRNA nearly as high as those produced by the pH 9 phenol treatment. The hot SDS-phenol procedure is also used for the extraction of polysomal mRNA? ,9 The relative efficiencies of the hot phenol and the cold pH 9 phenol treatments have not been compared on polysomal mRNA. Purification of mRNA

Binding Properties of Poly(A ) A variety of techniques are available for the selective absorption of poly(A)-containing RNA molecules and their effective separation from other types of RNA. In addition to its capacity to form complementary base-paired structures with poly(U) and poly(dT), the poly(A) segment is capable of binding to cellulose nitrate membrane filters (Millipore filters) at high ionic strength. 7 In this respect the behavior of poly(A) is similar to that of single-stranded DNA. Thus most of the labeled polysomal RNA from cells incubated in the presence of a low level of actinomycin will bind to Millipore in the presence of 0.5 M KC1. Ribosomal RNA is not retained under these conditions. Quantities of RNA up to 60 /~g have been adsorbed on a single filter. TM The maximum capacity of the filters has not been determined. The adsorbed RNA can be eluted with SDS in Tris buffer pH 9.0. The RNA cannot be adsorbed on the filters in the presence of the detergent. The unique behavior of poly(A) is not limited to binding to Millipore. As mentioned above, it will also bind to denatured proteins when the concentration of monovalent cation is sufficiently high. The precise conditions for selective binding to proteins, however, are not well understood, and this property does not appear to be useful for the purification of mRNA. With diluted suspensions of polysomes from mouse sarcoma 180 cells, the fractionation based on sequential phenol extraction with Tris buffers of pH 7.6 and 9.0 is quite effective (see Table I). With more concentrated polysome suspensions, the pH 9 fraction remains heavily contaminated with ribosomal RNA, and the mRNA is obtained in lower yields.12 The size of the poly(A) segment may play an important role in its 1TR. P. Perry, J. LaTorre, D. E. Kelley, and J. R. Greenberg, Biochim. Biophys. Acta 262, 220 (1972).

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THE ISOLATION OF MESSENGER R N A

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binding characteristics to Millipore. The newly formed mRNA of mammalian cells contains a poly(A) segment 100-200 nucleotides long. 1° Poly(A) of this size will bind effectively under the conditions described below. A substantially lower poly(A) size has been reported in the case of rabbit reticulocyte hemoglobin mRNA labeled for a long period of time? 8 It is possible that poly(A) segments with less than 50 nucleotides are not effectively retained on Millipore. 19 A substantial portion of the rabbit hemoglobin mRNA, however, is capable of binding to Millipore. '~ The procedures using complementary base pairing of the poly(A) appear to be effective with shorter polynucleotide chains. The Millipore-binding techniques provides a convenient analytical procedure for the routine assay of newly formed mRNA, since the adsorbed material can be counted directly on the filter. It permits the study of mRNA metabolism even in the absence of inhibitors of rRNA synthesis, since the labeled rRNA is not retained on the filters. This technique, however, does not appear to be effective for the selective adsorption of the nuclear poly(A)-containing RNA molecules. Procedure for the Adsorption of m R N A on Millipore Filters TM Reagents KC1 buffer: 0.5 M KCI, 1 mM MgClz, and 10 mM Tris.HC1, pH 7.6; SDS must be absent from solution Eluting buffer: 0.5% SDS in 0.1 M Tris.HC1, pH 9.0 Procedure. The adsorption can be carried out either in the cold or at room temperature. The latter condition appears to be more effective in reducing contamination by residual ribosomal RNA. The RNA solution is diluted 10- to 20-fold with KC1 buffer. The final RNA concentration should be no greater than 0.3 mg/ml. The solution is passed through a Millipore filter that had been presoaked for 30 minutes in KCI buffer. A rate of filtration of approximately 0.5 ml per minute is adequate. The filter is next washed several times with KC1 buffer. The filter with the adsorbed RNA is either dried for scintillation counting or used for elution of the adsorbed material as follows. The filter is placed in 0.5-1 ml of eluting buffer and kept in ice for about 30 minutes with occasional shaking. Because of the residual KC1 on the filter, a heavy precipitate of potassium dodecyl sulfate appears rapidly. This does not interfere with the elution of the RNA. The precipitate can be removed by centrifugation at 12,000 rpm for 10 minutes, and does not carry along any adsorbed RNA. 18H. Burr and J. B. Lingrel, Nature (London) New Biol. 233, 41 (1971). 19D. Sheiness and J. E. Darnell, personal communication.

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MESSENGERR N A AND PROTEIN SYNTHESIZING SYSTEMS

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The material recovered from the Millipore filters can be still contaminated with rRNA to an extent of approximately 50%. Zone sedimentation can be used to remove some of the contaminating RNA. O t h e r Separation Procedures for m R N A

Several procedures have been developed for the adsorption of mRNA using complementary base pairing. They involve coupling either poly(U) or p o l y ( d T ) to an insoluble matrix and adsorbing the R N A preparation under conditions that favor base pairing. P o l y ( U ) has been coupled to glass fiber filters by subjecting the filters coated with poly(U) to UV irradiationY ° The R N A solution is adsorbed to the treated filters in the presence of 0.12 M NaC1 in 10 mM Tris.HCl (pH 7.5). Poly(U)-glass fiber columns have been used in a similar fashion. P o l y ( d T ) coupled to cellulose is an effective adsorbent for p o l y ( A ) containing molecules. It had been used successfully for the isolation of hemoglobin m R N A on a relatively large scale. 21 The cyclohexylamine salt of thymidylic acid in pyridine is first polymerized in the presence of N,N'-dicyclohexylcarbodiimide (DCC) under strictly anhydrous conditions; the polymer is next coupled to thoroughly dehydrated cellulose, also in the presence of DCC. '-'-° The poly(dT)-cellulose is used as a small column, and the RNA solution is applied in the presence of 0.5 M KCI in 0.01 M Tris.HC1 pH 7.5. After thorough washing, the mRNA is eluted with 10 mM Tris, pH 7.5. zl It has been reported that untreated cellulose can adsorb p o l y ( A ) containing R N A under the conditions used for the poly(dT)-cellulose procedure, z3 These conditions are also similar to those for adsorption on Millipore filters. It is possible, however, that not all cellulose preparations exhibit this binding property. P o l y ( U ) coupled to agarose can be used as a selective adsorbent for poly(A)-containing R N A molecules. ~4 Also, messenger R N A hybridized with p o l y ( U ) can be isolated by chromatography on hydroxyapatite. 25 In this latter procedure, the complementary p o l y ( A ) - p o l y ( U ) segment on the RNA causes it to bind more tightly to hydroxyapatite. The mRNA, however, is recovered with contaminating p o l y ( U ) .

2oR. Sheldon, C. Jurale, and J. Kates, Proc. Nat. Acad. Sci. U.S. 69, 417 (1972). ~IH. Aviv and P. Leder, Proc. Nat..4cad. Sci. U.S. 69, 1408 (1972). ~2p. Gilham, g. Amer. Chem. Soc. 86, 4982 (1964). '~ P. A. Kitos, G. Saxon, and H. Amos, Biochem. Biophys. Res. Commun. 47, 1426 (1972). 54M. Adesnik, M. Salditt, W. Thomas, and J. E. Darnell, J. Mol. Biol. 71, 21 (1972). ~sj. R. Greenberg and R. P. Perry, J. Mol. Biol. 72, 3 (1972).