J. Mol.

BioZ. (1976) 106, 1061-1075

Purification of Globin Messenger RNA from Dimethylsulfoxide-induced Friend Cells and Detection of a Putative Globin Messenger RNA Precursor P. J. CURTIS

AND

C. WEISSMANN

Institut fiir Molekularbiologie I Universitlit Ziirich, 8049 Ziirich, Switzerland (Received

16 March

1976, and in revised form 24 June 1976)

Procedures are described that permit the detection and isolation of a specific messenger RNA as well as its precursor from total cell extracts. DNA complementary to the mRNA was elongated by the addition of dCMP residues and annealed with labeled cell RNA. The elongated DNA with RNA hybridized to it was isolated by chromatography on a poly(I)-Sephadex column. The method was used to isolate 32P-labeled globin mRNA from labeled ‘Friend cells, a mouse erythroleukaemic cell line, induced with dimethylsulfoxide to synthesize hemoglobin. 32P-labeled globin mRNA isolated by this procedure was estimated to be 80% pure by hybridization analysis and sedimented as a single peak at 10 S. Partial sequences were determined for 16 oligonucleotides derived from the 32P-labeled globin mRNA by RNAase T, digestion. The partial sequences purified for nine oligonucleotides corresponded to those predicted from the amino acid sequences of c( and p globin; the other oligonucleotides were presumably derived from non-translated regions. In order to detect a possible precursor to globin mRNA, RNA from induced Friend cells pulse-labeled with [32P]phosphate for 20 minutes was centrifuged through a sucrose gradient and the resulting fractions were analyzed for globinspecific sequences. Two peaks of globin-specific RNA were detected, a larger one at 10 S, the position of mature globin mRNA, and a smaller one at, 15 S.

1. Introduction It has been proposed by several authors (Attardi et al., 1966; Scherrer et al., 1966; Warner et al., 1966) that eukaryotic messenger RNAs are derived from precursor molecules of distinctly higher molecular weight than the mRNA itself. In the case of globin (Imaizumi et al., 1973; Spohr et al., 1974; Macnaughton et ab., 1974), these conclusions are based on the detection of globin mRNA-specific sequences in molecules substantially longer than about 600 to 700 nucleotides. In these experiments species were separated by sedimentation or electrophoresis, and the individual fractions were analyzed by hybridization with labeled globin complementary DNA. This approach allows the total content of globin-specific sequences to be determined and thus reflects the steady-state distribution of such molecules. In order to determine the precursor-product relationship between two RNA species, it is however desirable to carry out pulse and chase experiments which involve the determination of the labeled fraction of the globin-specific RNA population. Moreover, it would be of 69’

1061

1062

P. J. CURTIS

AND

C. WEISSMANN

interest to isolate the putative precursor molecule and to determine certain features of its structure. We have adapted the procedure of Coffin et al. (1974), which wasoriginally developed for the determination of labeled tumor virus-specific RNA, to the mouse globin system. In this paper we show that the method can be used to determine labeled globin-specific RNA at the 0.01% level. Moreover, the procedure was modified to allow the preparation of labeled 10 S globin mRNA of over 80% purity from Friend cells induced by dimethylsulfoxide to synthesize hemoglobin (Friend et al., 1971). Finally, evidence is provided for the existence of a 15 S RNA containing globinspecific sequenceswhich may represent the precursor of the 10 S globin mRNA.

2. Materials

and Methods

(a) Buffers The following buffers were used: Tris/EDTA buffer, 20 mM-Tris.HCl (pH 7*5), 0.5 XIIMEDTA; Tris/EDTA/SDSt buffer, Tris/EDTA buffer with 0.5% (w/v) SDS (recrystallized); NaCl/Tris buffer, 0.5 M-N&I, 10 mM-Tris.HCl (pH 7.5); NeCl/Tris/SDS buffer, NaCl/Tris buffer with 0.5% (w/v) SDS; SDS/formamide buffer, 90% (v/v) formamide (Fluka AG), 5% (v/v) 1 M-TriseHCl (pH 7.5), 5% (v/v) of 10% (w/v) SDS. (b) Cells Friend cells F707/M2 were a gift from Dr R. I. Freshney, the Beatson Institute for Cancer Research, Glasgow. Cells were grown in plastic Petri dishes (A/S Nuno, Roskilde, Denmark) in a 5% COP/air mixture at 3T°C, using supplemented Ham’s F12 medium containing 10% horse serum (Flow Laboratories or Gibco Biocult) (SFlS/lO%HS). The medium was prepared by dissolving IO.7 g Ham’s F 12 powder medium (Flow Laboratories), 24.8 ml MEM amino acids 50 x (Flow Laboratories), 2 mmol glutamine, lo6 units penicillin, 0.1 g streptomycin, 13.44 g NaHC03, 6 mmol NaOH in 900 ml water, adjusting the pH to 7.2 with COz, and adding 100 ml horse serum. Cells were maintained by diluting the cell suspension loo-fold into fresh medium every 4 to 5 days. For induction of hemoglobin synthesis, cells were plated at 2 x lo5 cells/ml of SFlB/lO%HS containing 1.5% Me,SO. The amount of hemoglobin was measured spectrophotometrically. Cells were suspended at 107/m.l in cold 5 mM-TrisvHCl (pH 7.5), 5 mM-NaCl, 0.75 mM-MgCl, and left in ice for 10min. The suspension was adjusted to 1 y. Nonidet 40 (Bender & Hobein), vortexed briefly and centrifuged for 5 min at 1300 g. The supernatant liquid was used to determine absorbance at 415 nm, using the value E&,, = 105 (Kabat et al., 1975). After 3 days the cells began to synthesize hemoglobin and gave a maximal yield after 5 days. At 5 to 7 days, the hemoglobin content was 0.02 mgj106 cells in freshly cloned cells. The response to Me,SO diminished during 2 to 3 months of continuous culturing; responsive cells were recovered by recloning. A cell suspension was diluted to 10 cells/ml in Ham’s F12 medium (Ham’s F12 powder medium with 1.2 g NaHCO$) and 10% horse serum: O*l-ml portions were pipetted into the wells of a microtest plate (Microtiter, DynEtech Produkte AG). After 5 to 7 days the contents of wells containing single colonies were transferred to plastic Petri dishes (5 cm diam.) containing 5 ml SF12/10%HS. It was noted that ttil clones that multiplied rapidly responded to Me,SO to give the amount of hemoglobin described above. (c)

Labeling

of cellular

RNA

Cells were labeled 3 to 4 days after the addition of 1.5% Me,SO. For [3H]RNA, 20 x lo6 cells were suspended in 10 ml SF12/10°kHS and O-5 mCi [5-3H]uridine (26 to 30 Ci/mmol; Radiochemical Centre, Amersham, Bucks) and incubated for 16 h at 37°C. For [3aP]RNA, phosphate-free SF12 was prepared according to Ham (1965) by omitting sodium phosphate t Abbreviations used: SDS, sodium dodeoyl sulfate; Me,SO, dimethylsulfoxide; plementary DNA.

oDNA, oom-

E’UTATIVE

GLOBIN

mRN.4

PRECURSOR

lWi3

and using horse serum dialyzed against 0.14 M-N&~, 50 mM-Tris (pH 7.5). A total of 2 x 10’ cells wassuspendedin lOmlphosphate-free SF12/10°/oHS containing up to 10mCi[32P]phosphate (carrier-free: Eidg. Institut fur Reaktorforschung, Wurenlingen, Switzerland) and incubated for 16 h at 37°C. For pulse-labeling with [32P]phosphate, 40 x lo6 cells were preincubated in 10 ml phosphate-free SF12/10°/cHS for 2 to 4 11, then 5 x lo6 cells were collected by centrifugation and resuspended in 0.25 ml phosphate-free SF12/100/,HS containing 50 mCi purified [32P]phosphate. For purification, carrier-free [32P]phosphat,e was mixed with 10 nmol unlabeled phosphate in 1 ml water, passed through Dowrx 50-H+ (4 cm x 0.5 cm column), adsorbed to Dowex l-Cl- (2 cm x 0.5 cm) and eluted with 0.2 N-HCl. The effluent was 3 times taken to dryness and redissolved in water. After mixing the phosphate with the medium the pH was checked and adjusted when necessary. The cell suspension was incubated for the time indicated (7 or 20 min) with cominuous shaking at 37°C. The cells were chilled, collected by centrifugation and further processed as described below, while the supernatant medium was transferred to a tube containing another batch of 5 x lo6 cells. The labeling procedure was carried for a total of 6 batches, after which over 60°& of the [32P]phosphate had been taken up. (d) Purification of cellular RAGi Cells were suspended in Tris/EDTA buffer at 2 x lo6 to 5 x lo6 cells/ml. The suspension was incubated at 37°C for 5 min in the presence of 2% SDS and 200 rg Pronase/ml (Calbiochem, Los Angeles, Calif.; predigested for 15 min at 37°C). The solution was extracted 4 times at room temperature with 1 vol. redistilled phenol; after each extraction, the phenol phase was rinsed with Tris/EDTA buffer. The aqueous phases were adjusted to 0.2 M-sodium acetate (pH 5.2) and the nucleic acids precipitated with 2.5 vol. ethanol. After dissolving and reprecipitating as above, the nucleic acids were dissolved in 0.1 ml water, adjusted to 50 mM-TrisHCl (pH 7*5), 10 mM-MgCla and incubated at 37°C for 5 min with 10 pg DNAase I (electrophoretically pure, Worthington Biochemical Corp., treat,ed with sodium iodoacetate by the procedure of Zimmermann & Sandeen (1966) to destroy residual RNAase). The solution was adjusted to 50 mM-EDTA and 0.5% SDS and extracted with 1 vol. phenol; after removing the aqueous phase, the phenol phase was rinsed with 50 ~1 Tris/EDTA buffer. The combined aqueous phases were passed through a Sephadex GlOO column (0.5 cm x 10 cm) with a 3-mm layer of Chelex-100 (Biorad; 100 to 200 mesh) in Tris/EDTA buffer. The excluded peak fractions of labeled RNA were pooled and the specific radioactivity was determined. [3H]RNA contained lo5 to 2 x lo5 cts/min per pg. [.32P]RNA labeled for 16 h contained lo6 to lo7 cts/min per rg and [32P]RNA labeled for 20 min contained 0-75x lo5 to 2 x lo5 cts/min per pg. Yeast RNA (British Drug Houses) was purified by dissolving in Tris/EDTA buffer containing 1% SDS and incubating at 37°C for 15 min with 200 rg Pronase/ml. The solution was extracted 6 times with 1 vol. phenol and RNA was precipitated with 2.5 vol. et,hanol. Partially degraded yeast RNA was prepared by heating purified yeast RNA (1 mg/ml) in 0.05 M-Na,CO, at 55°C for 60 min. The pH was adjusted to 7 and the solution passed through a 1 cm x 0.5 cm column of Chelex-100. Q/3 RNA was prepared as described by Weissmann et al. (1968) and mouse globin mRNA was kindly supplied by Dr R. Williamson, the Beatson Institute for Cancer Research, Glasgow, Scotland. 1251-labeled mouse globin mRNA was prepared according to Scherberg & Refetoff (1973). A solution containing (in 20 ~1) 1 rg mouse globin mRNh. 0.1 M-ammonium acetate (pH 5.0), 1 mM-thallium trichloride, 25 PM-potassium iodide and 10 to 20 &i sodium [1251]iodide (carrier-free, Eidg. Institut fiir Reaktorforschung, Wurenlingen, Switzerland) was heated at 66°C for 60 min. After cooling in ice, 5 pm01 TrisHCl (pH 8.9), 0.5 pmol sodium sulfite followed by 80 ~1 Tris/EDTA buffer were added. The reaction mixture was passed through a Sephadex GlOO column (0.5 cm h 10 cm) with a 3-mm layer of Chelex-100, in Tris/EDTA buffer. The iodinated RNA iri the excluded fractions was pooled and precipitated with ethanol. The precipitate was dissolved in 100 ~1 Tris/EDTA buffer, heated to 100°C for 45 s and centrifuged through a 5% to 23% sucrose gradient in 50 mM-TrisHCl (pH 7.5), 5 mM-EDTA, 0.1% SDS foi 2.5 h at 60,000 revs/min at 15°C in an SW65 Spinco rotor. The fractions corresponding to an 820,w value of 10 S were pooled and precipitated with ethanol. The spec. act. of iz51 labeled globin mRNA was lo6 to lo7 cts/min per pg.

1064

P. J. CURTIS (e) Synthesis

and

AND

elongation

C. WEISSMANN of globin-speci$c

DNA

Globin-specific DNA was prepared by oligo(dT)-primed synthesis with mouse globin mRNA (a mixture of a and fi globin mRNA) as a template for RNA-dependent DNA polymerase (purified from avian myeloblastosis virus by the method of Baltimore & Smoler, 1972). The specific activity of the enzyme was 25 units/pg protein, where 1 unit of enzyme is defined as 100 pmol dTMP incorporated in 15 min at 37°C with poly(A) as a template and oligo(dT),,.,, as primer. The synthesis was carried out in 4 ml of a mixture containing 40 pg mouse globin mRNA, 4 pg oligo(dT),,-,, (P.-L. Biochemicals, Milwaukee, Wise.), in 40 m&r-TrisHCl (pH 7.5), 30 mM-NaCl, 5 mM-MgCl,, 0.1 mM each of dATP, dCTP and dGTP, 0.1 mivr.[32P]dTTP (spec. act. 1000 cts/min per nmol) and 25 pg actinomycin/ml. After 60 min at 37°C the reaction was stopped by addition of SDS to a final concn of 0.5% and EDTA to 20 mM. The reaction mixture was extracted with 1 vol. phenol: the phenol phase was re-extracted with 0.5 vol. Tris/EDTA buffer and the combined aqueous phases were mixed with 2.5 vol. ethanol. The precipitated nucleic acids were dissolved in 0.2 ml 0.2 N-KOH and incubated at 37°C for 16 h. The solution was neutralized with HCl and passed through a Sephadex GIOO column (0.5 cm x 10 cm) in Tris/EDTA buffer. The labeled DNA in the excluded fractions was precipitated with 2.5 vol. ethanol. The yields ranged from 7 to 11 rg of labeled DNA. 1251-labeled globin mRNA was protected to 70 to lOOo/o from RNAase A and T, digestion after hybridization to a 2-fold (or greater) excess of this cDNA under the conditions described below (data not shown). The cDNA sedimented at 8 S in a neutral sucrose gradient. The cDNA was dissolved in the reaction mixture used for the elongation, to give a solution @al vol. 40 ~1) containing about 8 rg purified 32P-labeled globin-specific DNA, 2 mM-CoCl,, 125 m&r-potassium cacodylate (pH 7*0), 1 mm-dCTP, 0.2 mg bovine serum albumin/ml and 125 units terminal deoxynucleotidyl transferase/ml (P.-L. Biochemicals; spec. act. 6250 units/mg) and incubation was carried out at 37°C for 3 h. The reaction was terminated by the addition of SDS to a final concn of 0.5% and EDTA to 30 mM and the volume adjusted to 100 ~1 with Tris/EDTA/SDS buffer. The solution was extracted with phenol as described above and the combined aqueous phases were passed through a Sephadex GlOO column (0.5 cm x 10 cm) with a 3-mm layer of Chelex-100, in Tris/EDTA buffer. The labeled DNA was pooled from the excluded fractions. A portion of the poly(dC)-globin cDNA was passed through a poly(I)-Sephadex column under the conditions described below: 70 to 90% of the material was bound. (f ) Hybridization analysis RNA-DNA hybridization and detection of hybrid by (1) determining the resistance to RNAases A and T, or (2) absorption to poly(I)-Sephadex followed by treatment with RNAase were carried out as described by Coffin et al. (1974). The following quantities were routinely employed for a hybridization assay: 10 to 20 ng poly(dC)-globin cDNA, 1 pg oligo(C), 2 pg poly(U) and 0.5 to 5 pg labeled Friend cell RNA in 50 ~1 NaCl/Tris buffer. An internal control for the efficiency of hybridization, 1251-labeled mouse globin mRNA, was added to the hybridization mixture. When the unknown RNA was 32Plabeled, both ls51 and 32P radioactivities were determined on the same sample ; however, when the RNA was 3H-labeled, 2 samples were prepared, one with and another without the iodinated probe, in order to facilitate the counting of 3H radioactivity. In using 1251-labeled globin mRNA as a standard we assume that it is pure ; moreover, when it is used as internal standard, we assume that the cDNA is present in excess over globin RNA in regard to both a and /3 globin sequences. The purity of the 1251-labeled globin mRNA can be estimated from the finding that 70 to 100% of the radioactivity can be converted to RNAase resistance by hybridizing to a 2-fold excess of globin cDNA and that the globin cDNA does not hybridize detectably to non-induced Friend cell RNA. Poly(I)-Sephadex (binding capacity for poly(C), 60 pg/ml of bed volume) and oligo(C) were prepared as described by Coffin et al. ( 1974). (g)

Purification

of globin-apecijic

RNA

Labeled RNA was hybridized to poly(dC)-globin DNA hybrid was adsorbed to poly(I)-Sephadex, hybrid was eluted by SDS/formamide buffer.

from

induced

Friend

cells

cDNA in 60% formamide, the RNAand after washing the column, the

PUTATIVE

GLOBIN

mRNA

PRECURSOR

If Mi;,

The solutions used in the hybridization were passed through Chelex-100. Hybridizatiori was carried out in 100 ~1 of a solution containing 50% formamide, 0.75 ~-N&cl, 065 AITris.HCl (pH 7.5), 0.5% SDS, 5 mm-EDTA, 2 pg poly(U) (or 0.1% of the total RNA added), 16 pg oligo(C) (20 times the amount of poly(dC)-globin cDNA), ~200 pg labeled Friend cell RNA and 0.8 rg poly(dC)-globin cDNA (a P-fold excess over the measured content of globin-specific RNA). The solution was heated to 37°C for 4 11. Chromatographyon poly(I)-Sephadex was carried out at room temperature. The hybridization mixtum was diluted b-fold with NaCl/Tris/SDS buffer and applied to a poly(I)-Sephadex columrr 100 bed volumes of NaCl/Tris/ST)S which had previously been washed with approx. buffer. The amount of poly(I)-Sephadex was adjusted so that t’here was an excess of binding capacity for poly(C) over the amount of oligo(C) in the mixture. The column was washed with 100 bed volumes of NaCl/Tris/SDS buffer, followed by 5 bed volumes of Tris/SDS buffer. The hybridized RNA was eluted with 0.5 ml SDS/formamide buffer. of globin-specific RNA the hybridization and chromaTo obtain 80 to 90°’ ,. purification at this point the preparation contained some tography step was repeated. However, poly(I) which had been eluted from the poly(I)-Sephadex and this inhibited the rebinding of the RNA-DNA hybrid to poly(I)-Sephadex. The poly(1) could be removed by passage t,hrough poly(C)-Sephadex. However, it was necessary to do this in the presence of poly(U), since otherwise the poly(A) of the mRNA was crosslinked by poly(1) to the poly(C)-Sephadex. Poly(U) (5 pg) was added to the eluted RNA in 0.5 ml SDS/formamidc buffer, the solution was heated to 65°C for 2 min, diluted &fold with NaCl/TrisSDS buffer and passed through a poly(C)-Sephadex column. To the solution that flo\vccl t)hrorcgh, 200 pg partially degraded yeast RNA were added and the nucleic acids wer,~ precipitated with 2.5 vol. ethanol. The precipitate was dissolved in 0.5 ml Tris/ED’f:\ buffer, extracted with 1 vol. phenol as described above. and the combined aquec,us phases were passed through Chelex-100. The nucleic acids were precipitated with 2.5 vol. ethanol and dissolved in 100 ~1 of a solution containing 50yo formamide, 0.75 M-NaCl, 0.05 M-TrisHCl (pH 7.5), 0.5% SDS, 5 mM-EDTA, 2 pg poly(U), 32 pg oligo(CI), 0.8 pg poly(dC)-globin cDNA. Annealing and chromatography were carried out as described above. &!I RNA (20 pg) was added as carrier to the RNA eluted with SDS/formamide buffer. The solution was diluted 4-fold with NaCl/Tris/SDS buffer arid the nucleic ac*ids were prrcipitwkrd with 2.5 vol. ethanol. (h) Homochromatography The procedure is that of Volckaert et al. (1976). Labeled RNA was mixed wit11 Qg RNA as carrier to give a total of 20 rg RNA, precipitated with 2.5 vol. ethanol and dried. The precipitate was dissolved in 3 ~1 of 1 unit RNAase T,/$ in Tris/EDTA but&J and incubated at 37°C for 30 min. Analysis of T, oligonucleotides by RNAase A was carried out according to Adams et al. (1969) and the nucleotide composition of RNA&se A products was determined after enzymatic hydrolysis according to Hiramaru et al. (1966). (i)

Determination

of radioactivity

Acid-insoluble radioactivuy was determined by adjusting the solution to 0.25 w-N&:1, 50 mM-Tris.HCl (pH 7.5) and adding yeast RNA (final concn 200 &ml) and trichloroacet’ic acid (final concn 6?&, v/v). Formamide-containing solutions were first diluted to 50%) formamide. After 10 min at O”C, the precipitate was collected on Millipore filters (HAWP 304 FO, 25 mm diam.) and the radioactivity was determined in 10 ml of a s&ntillator solution containing 4 g butyl PBD (Ciba-Geigy, Basel) and 100 mg POP01 (Fluka AG, Buchs) per l/toluene. 1251 radioactivity was counted in a Nuclear Chicago gamma counter. The counting efficiencies were about 959; for 32P, 3076 for 3H and 467; for lZ51.

3. Results (a) Globin mRNA content of induced Friend cells Friend cells exposed to 14% Me,SO produced hemoglobin in detectable amounts after three days and contained quantities comparable to those found in normal red blood cells after five to seven days.

1966

P.

J.

CURTIS

AND

C. WEISSMANN

In order to obtain an estimate of their globin mRNA content, non-induced Friend cells and cells exposed to Me,SO for three days were labeled overnight with [3H]uridine. The RNA was extracted, purified and analyzed essentially as described by Coffin et al. (1974). The 3H-labeled RNA was annealed with poly(dC)-globin cDNA and the mixture passed through a poly(I)-Sephadex column which retained the poly(dC)-cDNA and any RNA hybridized to it. The column-bound material was then treated with RNAase A to remove non-hybridized RNA segmentsand to reduce the background, and the 3H-labeled RNA was eluted and quantified. Table 1 shows that in Me,SO-induced cells about 0.2% of the 3H-labeled RNA was globin-specific; no globin-specific RNA could be detected in uninduced cells (lessthan about O*O05°/0). We have obtained values as high as 0.5% after freshly cloning cells and as low as 0.03% after two to three months of continuous culture. Since no attempt was made to attain steady-state labeling conditions, these values do not necessarily reflect the absolute globin mRNA content of the cell RNA. In earlier studies with induced Friend cells, Preisler et al. (1973) obtained a value of 0.15% for the amount of globin mRNA using a labeled cDNA probe, and Gilmour et al. (1974) found 0.1 o/oglobin mRNA in cytoplasmic RNA, while Ross et aZ. (1974) found O*O3o/o. TABLE Content

Sourca of RNAt Uninduced cells Induced cells

Hybridization (sH]RNA W/min X 10-T 3.3 3.3 3.3 2.0 2.0 2.0

1

of globin-speci$cRNA of uninduced and dimethylsulfozide-induced Friend cells input 1251-labeled globin mRNA (cts/min x 10-s) 4.0 4.0

Radioactivity in hybrid (Oh of input) lZSI sH 0.007 0.007

(0.007)$ (0.007)

0.089 0.074

(0.017) (0.006)

-

Globin-speoific RNA § (% of total) 0 0

31.2(1.4)$ 0.23 0.22 33.07

(2.0)

t Cells were induced by addition of 1.6% Me&SO for 3 days. A total of 2 x 1Or cells (either uninduced or induced) were labeled with 0.5 mCi/O.S ml [3H]uridine in 10 ml SF12/10e!oHS for 16 h. Extracted RNA (spec. act. 3.5 x lo5 end 4.0 x lo5 ots/min per pg for RNA from uninduced globin mRNA (spec. act. 3.3 x 10s cts/min per and induced cells, respectively) and 1251-labeled pg) were annealed with 10 ng poly(dC)-globin cDNA and analyzed on poly(I)-Sephadex columns. Details are given in Materials and Methods. $ Background values (given in parentheses, not subtracted) were obtained by hybridizing parallel reaction mixtures in the presence of 0.1 pg globin mRNA. % [sH]hybrid - y. [3H]background ’ % Of tota1 = % _ % ’ loo’ 7 The [rz51]mRNA used as internal standard is usually recovered with a yield of about 40 to 50%. The losses are due to (1) incomplete hybridization (70 to loo%), (2) incomplete binding of the poly(dC)-cDNA to poly(I)-Sephadex (70 to 90%) and (3) incomplete elution from the poly(I)Sephadex (70 to 90%). [125I]hybrid

[126I]b~kgro~d

(b) PuriJicution of globin mRNA The isolation of globin mRNA was based on the same principle as the assay described above, namely annealing the RNA to poly(dC)-globin cDNA and isolating hybridized RNA by poly(I)-Sephadex chromatography. Since the treatment with RNAase had to be omitted, two cycles of hybridization and chromatography were

PUTATIVE

GLOBIN

mRNA

1067

PREC’URSOR

carried out’, and several modifications were introduced to reduce non-specific binding to the column. (1) First cycle of hybridization and poly(I)-Sephadex chromatography. In a typical experiment RNA extracted from Me&SO-induced Friend cells which had been labeled overnight with [32P]phosphate was hybridized wit,h an excess of poly(dC)globin cDNA. The poly(dC)-globin cDNA used for hybridization contains stretches of T residues (derived from the oligo(dT) used as primer for its synthesis and/or copied from the poly(A) segment of the globin mRNA) which could bind poly(A)conta,ining RNA. Moreover, (dC) residues can bind G-rich sequences of cellular RNA (Coffin et al., 1974). To prevent these interactions. poly(U) and oligo(C) were added to the mixture before annealing. Upon chromatography on poly(I)-Sephadex about 0.8% of the input radioactivity was retained and could be eluted with a formamide-containing buffer (cf. Table 2). The globin mRNA content of this fraction was about, 257; as assayed by hybridization. We have some evidence (unpublished data) that under our hybridization conditions ribosomal RNA molecules can form aggregates with each other and with globin mRNA. As t’his globin mRNA hybridizes to poly(dC)-globin cDNA, the entire complex can be retained on the poly(I)-Sephadex column. Fragmented ribosomal or yeast RNA added in sufficient excess over tho endogenous labeled RNA was found to largely prevent) the formation of t)hcse complexes. (2) Second cycle of hybridization and poly(I)-Sephadex chromatography. The material recovered from the first poly(I)-Sephadex chromatography contained small amounts of poly(1) bound to poly(dC)-globin cDNA. which was found to prevent attachment to poly(I)-Sephadex in a subsequent chromatography step. Most of the poly(I) could be removed by filtration through poly(C)-Sephadex. The labeled RNA was hybridized as before with a twofold excess of polp(dC)-globin cDNA. this t,ime in the presence of fragmented yeast RNA for the reasons set fort’h above. and the hybrid isolated on poly(I)-Sephadex. As shown in Table 2.094qb of the initial radioactive RNA was recovered, containing about 72:/, globin-specific sequences. The overall recovery of globin-specific RNA wa,s low in this experiment, a bout, 9T1. 111 the six purification experiments carried out so far the purit’y of the final preparation ranged from 68O/, to 95% (average, 80%) and overall recoveries were between 85°i, and 359;) (average 1976). (c) Characterization

of the purified

globin-speci$c

RMA

On centrifugation through a sucrose density-gradient the purified [a2P]RNA sedimented as a sharp peak at 10 S? as did authentic mature glohin mRNA purified from reticulocyte polysomes (Fig. 1). A comparison was made between the T, fingerprints of the purified [32P]RNA and total RNA extracted from induced cells labeled overnight with [3H]uridine. The t’wo RNAs were mixed, digested with RNAase T,, and the products separated by electrophoresis on cellulose acetate strips followed by homochromatography on PET plates (Volckaert et al., 1976). The PEI plate was first exposed to X-ray film to obtain an autoradiograph of the [32P]oligonucleotides and then impregnated with scintillator and a film exposed to reveal the 3H radioactivity. The autoradiograms (Fig. 2) show clearly that in the less densely populated areas of the plate several intense spots derived from 3H-labeled cell RNA (predominantly ribosomal RNA) are absent from the purified [32P]RNA. Two spots from the cell RNA overlapped w-it’li

cell

RNA

1st column 2nd column

0.80 0.036

0.035

100

(%)

Recovery

0.78

98

Total =P recovered (cts/min X 1O-G)

puri$cation

42.0

13.8

0.20

RNA from

induced

(5.5)

(Omi)

(0.037)

50.6

52.5

54.3

(1.6)

(1.1)

(1.0)

Hybridization assayS Radioactivity in hybrid (% of input) 1251 =P

of globin-speci$c

2 Friend

74.5

25.2

0.31

Globin-specific RNA (% of total)

cell RNA

66

100

RNA

The

(%)

8.6

Recovery globin-specific

t Cells were induced by 1.576 Me,SO for 3 days. A total of 10’ cells in 10 ml phosphate-free medium acre labeled with 2 mCi [32P]phosphate RNA (spec. act. 1.06 x 10s cts/min per pg) was extracted and purified as described in Materials and Methods. $ Analysis for globin-specific RNA was carried out as described in Table 1 and in Materials and Methods. The values arc the average of duplicate minations. Background values (given in parentheses, not subtracted) were obtained by hybridizing in the presence of 0.1 pg globin mRNA. The 32P-labeled RNA and ‘251-labeled globin mRNA (spec. act. lo6 cts/min par pg) was adjusted to give about 500 cts/min in the hybrid fraction.

RNA eluted from poly(I)-Sephadex RNA eluted from poly(I)-Sephadex

Induced

RNA?

Radiochemical

TABLE

16 h. deterinput of

for

of

PUTATIVE

0

GLOBIN

5

mRNA

IO Fraction

PRECURSOR

15

I Ofi!)

20

no.

FIG. 1. Sedimentation analysis of globin-specific [32P]RNA purified from induced Friend cells labeled for 16 h with [3sPJphosphate. The globin-specific [32P]RX.4 was purified as described in Mat,erials and Methods. A portion (3200 cts/min) was denatured at 100°C for 45 s in 100 ~1 Trial BDTA buffer containing 1 pg poly(A) and 4400 cts/min of globin [rZ”I]mRKA added as a marker. The sample was layered on 4.5 ml of a 5% to 230/o sucrose density-gradient in 50 mM-Tris.HCl (pH 7.5), 5 m&r-EDTA, 0.1% SDS and centrifuged for 2 h at 15°C and 60,000 revs/mm in an SW66 Spinco rotor. --o--o-, 32P radioactivity; -@-a-, la51 radioactivity. The arrows designate t.ho position of 28 S and 18 S ribosomal RNA run in a parallel gradient.

spots 5 and 7 of the purified globin RNA. The corresponding oligonucleotides, prepared from cellular [32P]RNA, gave different products on RNAase A and U, digestion compared with the products obtained from spots 5 and 7 (data not shown). For further characterization, the purified [32P]RNA was digested with T, RNAast% and the T, oligonucleotides were separated as before. The large oligonucleotides were eluted, further digested with RNAase A and the end products identified. The part’ial structures of 9 of 16 oligonucleotides examined were compatible with the amino acid sequences of tl and p globin (Table 3). Five oligonucleotides corresponded to ct globin, three to fl globin; and one oligonucleotide, no. 10, which occurred in a yield of t’vvo moles, could be accounted for by sequences common to both globins. These correlations appear all the more significant if one considers that no fits were possibln hetwrten a non-selected set of 30 large T, oligonucleotides from Qp RNA and thts ,$obin sequences (data not shown). (d) Detection of 15 X RNA containing globin mRNA sequ,ences Cells induced by growth in 1.5% Me,SO for 3 days were labeled for 16 hours by [:3H]uridine and for 20 minutes with [32P]phosphate. The RNA was extracted, heat denatjured for 45 secondsat 100°Cand sedimented through a sucrosedensit$y-gradient. Each fract’ion was assayed for globin-specific RNA by hybridization wit’h poly(dC)-

1070

P.

J.

CURTIS

AND

C. WEISSMANN

PUTATIVE

GLGBJX

mRNA

PHE~‘UKSOH

ICI71

globin cDNA and recovery of the hybrid by poly(I)-Sephadex chromatography and treatment with RNAase A. The result (Fig. 3) showed that 3H-labeled globinspecific RNA sedimented at 10 S, the position of mature mRNA. while 32P-labeled plobin-specific RNA sedimented as two peaks, a smaller one at 15 S and a larger one at 10 S. The RNA sedimenting to the bottom of the tube (fractions 1 and 2) was I’+ (bxamined for its content of globin sequences. Within the limits of our technique no such staquences could be detected (:_“...-..

(ye>

\

’

I

:.-. : -. _ .’

.-. - . . ‘6

,. -. --.” .. --. .#.* -. -1 ..’ -- ._.-.* **.-.. . - : .. . :- .:

,‘..

00

p,H 3.5

electrophoresis

-

Fro. 2. T, fingerprint analysis of globin-specific [32P]RNA. Globin-specific [3aP]RNA (40,000 ot,s/min) purified from induced Friend cells labeled for 16 h with [32P]phosphate was mixed with 2 x 10s cts/min (20 rg) of unfractionated [sH]RNA from induced Friend cells labeled for 16 h wit,h [“Hluridine and the mixture was digested with RNAase T, (0.15 units/pg) in 3 pl Tris/EDTA buffer for 30 min at 37’C. Fractionation of the products was by cellulose-acetate electrophoresis at pH 3.5 in the first dimension (where the products ran between 10 cm and 30 cm from the origin) followed by homochromatography on PEI plates in the second dimension. The plate was first autoradiographed for 32P radioactivity (a) and after 4 weeks impregnated with scintillator and autoradiographed for 3H radioactivity (b) (Randerath, 1970). A composite t,racing is shown in (c). Dark spots, 32P; light spots, 3H.

’

1.04 1.17 1.39 1.10 1.07

1.84 1.00

0.94

0.52 0.71 0.89 1.04 0.52 0.62 0.94 1.12

Relative molar yieldt structureS

(A-A-C, A-U, 3 C) A-A-G (2 A-C, 5 C, U) G It (2 A-C, 4 C, U) G (A-C, 3 C, U) A-A-G (A-C, 4 C) A-A-G

(A-A-A-C, 2 A-C, 8 C, 6 U) G (4 A-C, 8C, 9 U) G (A-C, 10 C, 8 U) G (2 A-C, 7 C, 2 U) A-A-G (A-A-C, 2 A-C, 5 C, U) A-G (A-A-A-U, A-C, C) A-A-A-A-G5 (A-A-A-U-, A-U, A-C, 3 C, 3 U) (A-U, A-C, 6 C, 4 U) G (A-A-C, 3 C, 4 U) A-A-G (A-U, A-C, 5 C, 2 U) G (A-A-U, A-C, 3 C, 2 U) A-A-G

Partial

G

C-A-A-C-A-U-C-A-A-G f Py-C-A-Py-C-A-Py-C-C-Py-G Py-C-A-Py-C-A-Py-Py-C-Py-G A-C-Py-U-C-Py-‘~-A-G C-C-C-A-C-A-A-G

A-U-U-U-Py-A-C-Py-C-C-Py-G

U-C-A-A-C-U-U-Py-A-A-G

U-A-C-U-U-Py-U-C-Py-U-C-Py-U-U-Py-G U-U-Py-C-Py-C-A-C-C-A-C-C-A-A-G A-C-U-A-A-C-C’-A-Py-Py-U-A-G

of corresponding deduced from amino sequence7

sequence

z and /I polypeptides

Possible oligonucleotides

of T, productr with globin T, acid

a . B I a a /I a fi

116120 121-125 &ll 112-115 117-120 88-90 142-145

a 96-99

/I 41-46 a 36-40 /3 75-79

Location sequence globin

of in

Friend cells were induced by the addition of 1.5% Me,SO. 4.5~ lo7 cells in 15 ml phosphate-free SF12/10%HS were labeled with 20 mCi [32P]phosphate for 16 h. The extracted RNA (380 pg, specific 32P radioactivity 7 x lo6 cts/min per pg) was purified as described in Materials and Methods, precipitated with ethanol and dissolved in 100 ~1 Tris/EDTA buffer. The sample was heated for 45 s at 100°C in the presence of 1 pg poly(A) and centrifuged through a sucrose gradient for 2.5 h, as described in the legend to Fig. 1. The 10 S region (4.6 x lo5 cts/min) was pooled, 20 pg Qfi RNA were added as carrier, and the RNA was precipitated with ethanol. Digestion with RNAase T, and separation of the digestion products were carried out as described in the legend to Fig. 2. The oligonucleotides designated by numbers in Fig. 2(c) were eluted. dige3t,ed with RNAase A, and the resulting products characterized as described in Materials and Methods. t The average radioactivity per phosphate was determined from all oligonucleotides listed. The relative molar yield of an oligonuoleotide was calculated as (radioactivity in oligonucleotide/average radioactivity per phosphate) x number of phosphates in the oligonuoleotide. $ Deduced from RNAase A digestion products. The quantitation of the products was accurate to i20%. 5 Presumed sequence on basis of electrophoretic mobility only. 7 Taken from Dayhoff (1972). Nuoleotide sequences were matched to protein sequences by trial and error.

1 2 3 4 6 6 7 8 9 10 11 12 13 14 15 16

Product

Correlation

TAZLE 3

PUTATIVE

GLOBIN

mRNA

PRECURSOR

1073

FIG. 3. Sucrose gradient-centrifugation of labeled Friend cell RNA. Location of globin-specific sequences by hybridization. Induced cells were labeled with [ssP]phosphate for 20 min. The 3H radioactivity 1.2 x 10s cts/min per pg; specific szP radio. extracted RNA (676 ag, specific activity 2.8 x 10s cts/min per pg) was denatured at 100°C for 46 s in 100 ~1 Tris/EDTA buffer and centrifuged as described in the legend to Fig. 1 for 16 h at 15°C and 32,000 revs/min in an SW41 Spinco rotor. From each fraction (360 /.d), 16 ~1 was hybridized with 20 ng poly(dC)-globin cDNA and assayed by the poly(I)-Sephadex method. Total [32P]RNA, ---u--n--; hybridized C3sP]. RNA, -O-O-; hybridized [sH]RNA, -@-a-.

4. Discussion The technique of hybridizing globin-specific RNA to poly(dC)-globin cDNA and isolating the complex on poly(I)-Sephadex has been used to purify labeled globin mRNA. The RNAase treatment of the column-bound complex, which in the analytical procedure ensured low levels of non-specific binding, was replaced by an elution schedule involving low ionic strength buffer. One round of hybridization and poly(I)Sephadex chromatography yielded a product that was 25% globin RNA. The main contaminant, which appeared to be ribosomal RNA, was effectively removed in a second round of hybridization, carried out in the presence of partially degraded yeast RNA as a competitor. It was not feasible to add sufficient partially degraded yeast RNA to eliminate the labeled ribosomal RNA in the first round of hybridization, since large quantities of yeast RNA interfered with the subsequent poly(I)-Sephadex chromatography. The RNA purified by two rounds of hybridization contained on average 807; globin RNA as assayed by hybridization. The purification could also be carried out using the more readily accessible rabbit poly(dC)-globin cDNA in the hybridization step ; a similar degree of purification was attained (data not shown). The purified RNA sedimented in a sucrose gradient at 10 S with the same profile as 1251-labeled mouse globin mRNA and showed no evidence of degradation. WC! have also observed that the sedimentation profile of 45 S preribosomal RNA which had been subjected to hybridization and poly(I)-Sephadex chromatography showed little change, indicating that the isolation procedure causes little degradation of RNA (unpublished results).

1074

P.

J.

CURTIS

AND

C. WEISSMANN

The fingerprint of large T, oligonucleotides derived from the purified globin RNA did not contain detectable quantities of large T, oligonucleotides characteristic for total cell RNA. Partial sequences were established for 16 large T, oligonucleotides isolated from the purified globin RNA. Nine of these were compatible with sequences predicted from the amino acid sequences of u and /3 mouse globin. This is as expected, since the globin cDNA used in the isolation procedure was transcribed from a mixture of cc and /3 globin mRNA. One of 16 oligonucleotides (no. 5) showed questionable fit and six showed no fit with sequences deduced from the amino acid sequences. The ratio of S/l6 corresponds approximately to the ratio of non-coding region to total length of mRNA (Morrison et al., 1974). The chemical and hybridization analyses thus show that the labeled RNA purified is globin-specific and contains no detectable, defined contaminants. Evidence has been presented earlier for the existence of a precursor to globin mRNA (Imaizumi et al., 1973; Spohr et al., 1974; Macnaughton et al., 1974). Labeled cDNA was used as a probe to detect the presence of globin-specific sequences in RNA molecules larger than 10 S in nuclear RNA from duck erythroblasts. By this approach the steady-state composition of nuclear RNA could be examined, but neither pulse and chase experiments nor further characterization of the putative precursor were possible. Using our hybridization procedure we have examined the RNA of cells that had been labeled for a long period with [3H]uridine and then subjected to a pulse of [32P]phosphate. The RNA was sedimented through a sucrose gradient and the globin-specific radioactive RNA was assayed for in each fraction. In a 20-minute pulse with [32P]phosphate two well-resolved peaks of globin-specific [32P]RNA, a smaller one comprising about 20% of the globin-specific RNA sedimented at 15 S, the larger one at 10 S (coincident with the globin-specific [3H]RNA) were identified. In experiments to be reported on elsewhere, we have found that on [32P]RNA sedimented shortening the pulse to seven minutes, the globin-specific predominantly in the 15 S region. This would be in keeping with the assumption that a globin mRNA precursor with a sedimentation coefficient of 15 S is processed to the mature 10 S form. More detailed experiments are of course required to substantiate the precursor-product relationship and to obtain a reliable estimate of the transition times. The possibility that the 15 S RNA is an aggregate of 10 S RNA is very unlikely for two reasons. (1) The heat treatment to which the sample is subjected before sucrose gradient centrifugation (100°C for 45 seconds) is more stringent than that recommended by MeKnight & Schimke (1974) and suffices to denature doublestranded Qp RNA (unpublished results; see also Flavell et al., 1974). (2) The 3Hlabeled, endogenous 10 S RNA which serves as an ideal internal reference, shows no evidence of aggregation. In a preliminary experiment, globin-specific 15 S RNA has been isolated by our hybridization procedure in combination with sucrose density-gradient centrifugation. It should thus become possible to characterize the putative precursor in regard to its content of globin mRNA-like sequences, the nature and position of the additional sequences, and its termini. We are very grateful to Dr R. I. Freshney for providing Dr R. Williamson for mouse globin mRNA. We also thank us with oligo(C) and poly(I)-Sephadex and for many helpful supported by the Schweizerische Nationalfonds.

us with Friend cells and Dr L. Cashion for supplying discussions. This project was

PUT,1TIVE

GLOBIN

mRNA

PREC’UHSOR

1075

REFERENCES Adams, J. M., Jeppesen, P. G. N., Sanger, F. & Barrell, B. G. (1969). Nature (London), 223, 1009-1014. Attardi, G., Parnas, H., Hwang, M.-I. H. dt Attardi, B. (1966). J. 1MoZ. Biol. 20, 145-182. Baltimore, D. & Smoler, D. F. (1972). J. Biol. Chem. 247, 7282-7287. (‘offin, J. M., Parsons, J. T., Rymo, L., Haroz, R. K. 8: Weissmann, C’. (1974). J. .Vol. Biol. 86, 373-386. Dayhoff, M. A. (1972). Editor of Atlas of Protein Sequence and Structure, vol. 5. Flavell, It. A,, Sabo, D. L., Ba,ndle, E. F. & Weissmann, C. (1974). J. ,VoZ. Biol. 89, 265272. I’riend, C., Seller, W., Holland, J. G. & Sato, T. (1971). Proc. Nat. ,4cad. Sci., U.S.A. 68, 378-382. (iilmour, R. S., Harrison, P. R., Windass, J. D., ,4ffara, N. A. & Paul, J. (1974). Cell uiff. 3, 9-22. Ham, R. G. (1965). Proc. Nut. Acad. Sci., U.S.A. 53, 288-293. Hiramaru, M., Uchida, T. & Egami, F. (1966). Anal. Biochem. 17, 135-142. lmaiznmi. T., Diggelmann, H. & Scherrer, K. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 1122-1126. Kabal, D., Sherton, C. C., Evans, L. H., Bigley, R. & Koler, K. D. (1975). CeZZ, 5, 331.-338. Macnaughton, M., Freeman, K. B. & Bishop, J. 0. (1974). Cell, 1, 117-125. MeKnight, 0. S. & Schimke, R. T. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 4327-4331. Morrison, M. R., Brinkley, S. A., Gorski, J. & Lingrel, J. B. (1974). J. Biol. Chem. 249, 5290-5295. Preisltlr, H. D., Houseman, D., &her, W. & Friend, C. (1973). Proc. Nut. Acad. SC;., 1 ‘.S.A. 70, 2956-2959. Randf~rat~h, K. (1970). Anal. Biochem. 34, 188-205. Ross, J., Gielen, J., Packman, S., Ikawa, Y. & Leder, P. (1974). J. Mol. Biol. 87, 697.-714. Scherberg, N. H. & Refetoff, S. (1973). Nature New Biol. 242, 142-145. Scherrer, K., Marcaud, L., Zadjela, F., London, I. M. & Gros, F. (1966). Proc. Xnt. Acad. q: k~li., P.S.A. 56, 1571-1578. Spohr, G., Imaizumi, T. & Scherrer, K. (1974). Proc. Nat. Acad. Sci., U.S.A. 71,5009-5013. Volckaert, G., Min Jou, W. & Fiers, W. (1976). Anal. Biochem. 72, 433-446. Warnc:r, J. R., Sooiro, R., Birnboim, H. C., Girard, M. & Darnell, J. E. (1966). J. Mol. 11iol. 19, 349-361. Weissmann, C., Colthart, L. & Libonati, M. (1968). Biochemistry, 7, 865-874. Zimmermnnn, S. B. & Sandeen, G. (1966). Anal. Biochem. 14, 269-277.